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Winter 2015
Instrumentation and development of a massspectrometry system for the study of gas-phasebiomolecular ion reactionsZiqing LinPurdue University
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Recommended CitationLin, Ziqing, "Instrumentation and development of a mass spectrometry system for the study of gas-phase biomolecular ion reactions"(2015). Open Access Dissertations. 503.https://docs.lib.purdue.edu/open_access_dissertations/503
INSTRUMENTATION AND DEVELOPMENT OF A MASS SPECTROMETRY
SYSTEM FOR THE STUDY OF GAS-PHASE BIOMOLECULAR ION REACTIONS
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Ziqing Lin
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
May 2015
Purdue University
West Lafayette, Indiana
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ACKNOWLEDGEMENTS
I am deeply thankful to my PhD advisor, Prof. Zheng Ouyang. He is more than a
mentor or supervisor, but a kind friend, giving me a fantastic PhD experience at Purdue.
His passion, courage and extraordinary vision in scientific research makes him an
outstanding scientist and engineer. Prof. Ouyang works hard day and night, while on the
other side providing a free and comfortable environment for me and other colleagues in
the group to do research and raise opinions without pressure. These precious
characteristics no doubt affect me in my professional life. When we first met in Tsinghua,
he told me that PhD life is a best opportunity to test our boundary of capabilities. I learnt
a lot during my PhD study, not only in terms of technical knowledge but the
determination and belief in solving a problem. Five years is not a short period, I truly
appreciate his supervision and encouragement for me to explore the scientific world and
myself. It is my honor to know Zheng as a person and have the opportunity to work with
each other for both research and teaching experiences. I sincerely wish him good luck for
his future endeavors.
I would also like to extend my thanks to Prof. Yu Xia in Department of Chemistry. I
received a lot of valuable suggestions and comments from her for gas-phase ion
chemistry. Prof. Xia is very sophisticated and precise about her scientific findings. Her
dedication and persistence shows me how one could possibly be devoted to her career.
Moreover, Dr. Xia has a charismatic personality and always welcomes discussions at any
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time. She also allowed me to use the nanospray tip puller and the commercial mass
spectrometer in her laboratory, which was a great favor during my PhD study.
It is my privilege to have Prof. Kinam Park and Prof. Corey Neu in my thesis
committee, who are always trying to do everything to help. Prof. R. Graham Cooks,
although not in my committee, has offered tremendous guidance and support in different
perspectives of my graduate studies. I would also like to thank Prof. Mingji Dai, Dr.
Yang Yang from Purdue University, Prof. František Tureček from University of
Washington, Prof. Barney Ellison, Prof. Veronica M. Bierbaum from University of
Colorado, Prof. Michael L. Gross from Washington University, and Dr. Amber L. Russell
from Badger Technical Services for their kind help in organic synthesis, theoretical
calculation, and tips for the pyrolysis nozzle.
I am indebted to all the group members and alumni in Prof. Ouyang, Xia and Cooks’
research group. It has been a wonderful experience to have such a close relationship with
so many people. Dr. Tsung-Chi Chen, Dr. Wei Xu, Chien-Hsun Chen and Linfan Li are
the colleagues who helped me to build my instrument. Jason Duncan was a technician in
Cooks’ group, always delivering solid supports when I asked for different electronic
controls to achieve varies functions. Lei Tan is a close collaborator in Xia’s group with
whom I worked together on gas-phase ion chemistry. Besides, it is always inspiring and
pleasant to discuss with group members such as Dr. He Wang, Dr. Qian Yang, Dr.
Sandilya Garimella, Dr. Xiaoyu Zhou, Xiao Wang, Yue Ren, Melodie Du, Yuan Su, etc.
Without all your help, I would not have been able to finish my PhD program.
I would also like to take the time to acknowledge my parents who have been
supporting me mentally and financially throughout my student life in China and my PhD
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abroad. They have been extremely patient and understanding and I would like to thank
them for all the amazing opportunities they have given me over the years. Last but not
least, I owe my deepest gratitude to my lovely girlfriend and dance partner Esther Foo,
who shares with me tears and laughter, sadness and joy in my life and for being
supportive throughout this journey. Having blessed with a strong memory where I could
recall minute details in my everyday life, I’m glad that I would have the chance to
remember all the wonderful moments in my PhD life, and for this, I am eternally grateful.
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TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
ABBREVIATIONS .......................................................................................................... xv
ABSTRACT…. .............................................................................................................. xviii
CHAPTER 1. INTRODUCTION ..................................................................................... 1
1.1 Gas-phase biomolecular ion reactions and mass spectrometry
instrumentations .................................................................................................................. 1
1.1.1 Tandem mass spectrometry in gas-phase ion reactions .......................5
1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure
7
1.1.3 Instrumentations for gas-phase ion reactions in vacuum ...................13
1.2 Conclusion ...............................................................................................38
1.3 References ................................................................................................40
CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME-
BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE
ION/MOLECULE AND ION/ION REACTIONS ........................................................... 50
2.1 Introduction ..............................................................................................50
2.2 Instrumentation ........................................................................................52
2.3 Materials ...................................................................................................54
2.4 Results and discussion .............................................................................54
2.4.1 System configuration .........................................................................54
2.4.2 Pressure effect on gas-phase ion reactions ........................................59
2.4.3 Dynamic gas flow effect on gas-phase ion reactions .........................62
2.5 Conclusion ...............................................................................................69
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Page
2.6 References ................................................................................................70
CHAPTER 3. Gas-Phase Reactions of C3H2 with Protonated Alkyl Amines: formation
of a c-n covalent bond ....................................................................................................... 73
3.1 Introduction ..............................................................................................73
3.2 Instrumentation ........................................................................................74
3.3 Materials ...................................................................................................78
3.4 Results and discussion .............................................................................79
3.4.1 Carbene reaction with protonated alkylamines ..................................79
3.4.2 Theoretical calculation of the reaction mechanism ...........................83
3.4.3 Experimental evidences of the reaction mechanism ..........................86
3.5 Conclusion ...............................................................................................91
3.6 Appendix ..................................................................................................92
3.7 References ..............................................................................................106
CHAPTER 4. Gas-Phase c-C3H2 carbene reactions with biomolecular ions ............... 109
4.1 Introduction ............................................................................................109
4.2 Experimental section ..............................................................................110
4.3 Results and discussion ...........................................................................110
4.3.1 Nucleobases and nucleosides ...........................................................110
4.3.2 Amino acids, peptides, proteins, and lipids .....................................117
4.4 Conclusion .............................................................................................131
4.5 References ..............................................................................................132
VITA…………. .............................................................................................................. 134
PUBLICATIONS ............................................................................................................ 136
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LIST OF TABLES
Table ..................................................................................................................... Page
Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics ................... 3
Table 3.1 Ammonium reactions with various radical precursors ..................................... 92
Table 3.2 MS3-MS5 CID spectra of selected reaction products ........................................ 97
Table 3.3 GDEI spectra of radical precursors ................................................................. 101
Table 3.4 Ion/molecule reactions of c-C3H3+ and neutral amines .................................. 104
Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4
...................................................................................................................... 111
Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 ...................... 115
Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 .............. 126
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LIST OF FIGURES
Figure ..................................................................................................................... Page
Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase ....................... 2
Figure 1.2 Tandem mass spectrometry in space vs. in time ............................................... 5
Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low
temperature plasma (LTP)35 or ultraviolet (UV) lamp36. ................................. 7
Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to
form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI
plume: (a) peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of
AGC(TFTSC)K.42 ............................................................................................ 9
Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free
thiol43 and (b) a disulfide bond44 .................................................................... 10
Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with
a subsequent MS2 scan to improve sequence information of disulfide
peptides5, 35 ..................................................................................................... 11
Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin
together with possible retro P-B reactions.52 ................................................. 12
Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS.
MS1 spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS2
CID of the P-B reaction products at (c) m/z 339.3; and (d) structures of the
diagnostics ions (structures 3 and 5). The structures explain the origin of the
26-Da mass difference.52 ................................................................................ 13
Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with
collision gas for chemical mixture analysis6a ................................................ 15
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Figure ..................................................................................................................... Page
Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56 .......... 16
Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59 ................... 17
Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3
dimensions. A fixed mass (filled circle) or variable masses (open circle) can
be chosen for reactions.61 ............................................................................... 18
Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum
for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates.
The angled Q1 axis displays the precursors of m/z 43 ions, whereas the
horizontal Q5 axis displays the ion/molecule products of these CID-generated
m/z 43 ions arising from reactions with isoprene. Triple-stage sequential
precursor spectra extracted from the 3D familial spectrum by selection in Q5
of the final products of (b) m/z 81 and (c) m/z 111.63 .................................... 19
Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled
with a radical source for ion/radical reactions69 ............................................ 21
Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and
back), two opposite excitation plates (right and left), and two detection plates
(top and bottom). Ions enter the cell through the front trapping plate. Mass
spectrum generated by fast Fourier transformation.74 .................................... 23
Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78 ............. 24
Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84 ........................................ 24
Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T
instrument.89 ................................................................................................... 25
Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed
valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions.
........................................................................................................................ 27
Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and
differential pumping. LQIT1 and LQIT2 are the first and second linear
quadrupole ion traps (LQIT).91 ...................................................................... 28
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Figure ..................................................................................................................... Page
Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two
electrospray ionization (ESI) sources and one atmospheric sampling glow
discharge ionization (ASGDI) source interfaced.98b ...................................... 30
Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an
atmospheric sampling glow discharge ionization source on the side of Q3
linear ion trap for ion/ion reactions.100 ........................................................... 31
Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB)
gun for metastable atom-activation dissociation19 and (b) a laser coupled
linear ion trap for infrared multiple photon dissociation (IRMPD)102b and
ultraviolet photon dissociation (UVPD)103. ................................................... 32
Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer
for ion/ion reactions with dual ion source.104 ................................................. 34
Figure 1.25 Schematic of an Orbitrap Elite.107a ................................................................ 35
Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with
differential pumping system and ion optics and (b) miniature DAPI-RIT mass
spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini
12 of Point-of-care personal mass spectrometer.110, 113 .................................. 36
Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b)
Manifold pressure measured during scanning, with an open time of 20 ms and
a closed time of 850 ms for DAPI.110 ............................................................ 37
Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis
radical source for the study of gas-phase ion/radical reactions. .................... 39
Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General
waveforms of the in-trap gas-phase reactions ................................................ 55
Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide ............... 56
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Figure ..................................................................................................................... Page
Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with
deprotonated pentachlorophenol anions (insert: Isolation of the triply charged
species); (b) MS spectrum of Angiotensin I without opening DAPI-2; (c) MS
spectrum of isolated triply charged peptide ions with DAPI-2 opening but the
nanoESI source being turned off for anion production; and (d) aveforms of
the ion/ion reaction (dc was set to -10 V during mass analysis). ................... 58
Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different
pressures. Activation time 30 ms, amplitude 200 mV. .................................. 60
Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6
mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay,
from the DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and
blue columns illustrate the positions of ion introduction, dipolar ac excitation,
and MS scan, respectively; (d) Ion-trap CID spectrum of m/z 195 on DAPI-
RIT-DAPI system; (e) Ion-trap CID spectrum of deprotonated 9-
anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule reactions of
deprotonated anthracene ions with water vapor on QTRAP 4000. The CID
activation time was set to 30 ms on DAPI-RIT-DAPI system. ..................... 62
Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b)
Internal energy distributions of DAPI vs. continuous API for ion introduction;
Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms
with capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the
fragment of p-R (R = OCH3, CH3, Cl, CN, NO2). ......................................... 64
Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a)
MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by
CID of protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals
and dimethyl disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25
ms; q=0.35 for thiyl radical ions); (d) Ion/molecule reactions between isolated
m/z 307 from panel (c) with dimethyl disulfide. ............................................ 66
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Figure ..................................................................................................................... Page
Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times.
........................................................................................................................ 67
Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V;
(b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions. ............ 68
Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical
species and (b) the general MS scan function for ion/radical reactions (y-axis:
voltage). DAPI: discontinuous atmospheric pres-sure interface; RIT:
rectilinear ion trap; GDEI: glow discharge electron impact; rf: radio
frequency; ac: alternative current. .................................................................. 75
Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with
heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2
reaction of protonated n-heptylamine with heated d12-pentane; and (d) MS2
reaction of protonated n-heptylamine 18C6 complex with heated 1,3-
dibromopropyne. ............................................................................................ 79
Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b)
propargylbromide, (c) 1,5-hexadiene, and (d) pentane. ................................. 80
Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c)
cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID
spectra of the reaction products for (d) dibutylamine and (e) tributylamine. 81
Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester
with heated allylchloride, and their corresponding product MS3 CID spectra
(c) and (d), respectively. ................................................................................ 82
Figure 3.6 Calculated PES of (a) CH3NH3+/c-C3H2 reaction to form a C-N covalent bond
from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ,
kJ/mol) and (b) the two Brauman intermediates as a function of the distance
of N-H---C, blue square: CCSD(T)/aug-cc-pVTZ single point energies on
M06-2X/6-311+G(2d,p) optimized geometries; open circle: M06-2X/6-
311+G(2d,p) optimized; filled circle: B3LYP/6-311+G(2d,p) optimized. .... 85
Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A ...... 86
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Figure ..................................................................................................................... Page
Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional
activation: relative intensity of parent complex ([C], blue circle), covalent
product ([C-40], red diamond), proton-transfer product (m/z 39, green
triangle), and direct dissociation ([C-38], green square) as a function of
activation energy. ........................................................................................... 87
Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b)
ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and
(e) %Covalent of protonated alkyl amines/c-C3H2 reactions as a function of
GBs of the corresponding alkyl amines. The %Covalent is calculated as I[c-
40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40], and I[c-38] correspond to the
intensity of m/z 39 as the proton-transfer product, neutral loss of 40 Da as the
covalent product, and neutral loss of 38 Da as the direct-dissociation product,
respectively, in the MS3 CID spectra based on 80-90% parent ions
fragmented. Error bars were based on 5 duplicates of an average of 3 spectra.
........................................................................................................................ 90
Figure 3.10 MS3 reaction product CID spectrum of C3H3+and neutral n-heptyl amine ... 91
Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated
adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d)
adenosine carbene monoadduct ................................................................... 113
Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated
uridine; and MS3 CID spectrum (c) uridine carbene monoadduct ............... 114
Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum
of Lys/carbene reaction product, and (c) proposed fragmentation pathway of
the reaction product. ..................................................................................... 119
Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged
MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d)
MRFA/carbene reaction product. ................................................................. 120
Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride
at (a) 750, (b) 800, and (c) 900 degree Celsius. ........................................... 122
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Figure ..................................................................................................................... Page
Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its
reaction with heated allylchoride. ................................................................ 123
Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated
cystine, and (c) singly charged oxidized glutathione. .................................. 124
Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double
bond containing LPC (18:2) sodium adduct. No reaction product was
observed. ...................................................................................................... 125
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ABBREVIATIONS
ac alternative-current
APCI atmospheric pressure chemical ionization
API atmospheric pressure interface
ASGDI atmospheric sampling glow discharge ionization
B magnetic field
BIRD blackbody infrared dissociation
c-C3H2 cyclopropenylidene
C3H2 vinylidene carbene
CAD collision-activated dissociation
CI chemical ionization
CID collision-induced dissociation
DAPI discontinuous atmospheric pressure interface
dc direct-current
E electric field
ECD electron capture dissociation
EDD electron detachment dissociation
EI electron impact
ESI electrospray ionization
ETD electron transfer dissociation
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FA flowing-afterglow
FAB fast atom bombardment
FT-ICR Fourier transfer ion-cyclotron-resonance
GB gas-phase basicity
GDEI glow discharge electron impact
GIB guided ion beam
H/D hydrogen/deuterium exchange
IR infrared
IRMPD infrared multiphoton dissociation
ISM interstellar medium
LIAD laser-induced acoustic desorption
LIT linear ion trap
LMCO low mass cut-off
LQIT linear quadrupole ion trap
LTP low temperature plasma
MAD metastable atom-activated dissociation
MALDI matrix-assisted laser desorption ionization
MIKES mass-analyzed ion-kinetic-energy spectrometry
MRM multiple reaction monitoring
MS mass spectrometry
MSn tandem mass spectrometry
OH hydroxyl radical
PD photon dissociation
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ppm parts per million
pptv parts per trillion volume
PTM posttranslational modification
QqQ triple-quadrupole
QqQqQ penta-quadrupole
rf radio frequency
RIT rectilinear ion trap
ROS reactive oxygen species
SIFT selected-ion flowing-tube
SORI sustained off-resonance irradiation
SRM selected reaction monitoring
TOF time-of-flight
UV ultraviolet
UVPD ultraviolet photon dissociation
VOC volatile organic compound
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ABSTRACT
Lin, Ziqing. Ph.D., Purdue University, May 2015. Instrumentation and Development of a
Mass Spectrometry System for the Study of Gas-Phase Biomolecular Ion Reactions.
Major Professor: Zheng Ouyang.
Gas-phase reactions of biomolecular ions are highly relevant to the understanding of
structures and functions of the biomolecules. Mass spectrometry is a powerful tool in
investigating gas-phase ion chemistry. Various mass spectrometers have been developed
to explore ion/molecule reactions, ion/ion reactions, ion/photon reactions, ion/radical
reactions etc., both at atmospheric pressure and in vacuum. In-vacuum reactions have an
advantage of involving pre-selecting the ions for the reactions using a mass analyzer.
Over the decades, a variety of mass analyzers have been employed in the research of ion
chemistry. Hybrid configurations, such as quadrupole ion trap with a time-of-flight and or
a quadrupole ion trap tandem with an Orbitrap, have been utilized to improve the
performances for both the reaction (in trapping mode) and the mass analysis (accurate
mass measurements).
Complicated instrument structures, including ion optics, multiple mass analyzers and
differential pumping for high vacuum, are typically required for the mass spectrometers
for gas phase ion chemistry study. An alternative approach is to simplify the
instrumentation by using pulsed discontinuous atmospheric pressure interfaces for
introducing ionic or neutral reactants and a single ion trap as both the reactor and the
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mass analyzer. Such a simple mass spectrometry system was set up and demonstrated
using two discontinuous atmospheric pressure interfaces in the study for this thesis. It
was capable of carrying out ion/molecule and ion/ion reactions at an elevated pressure
without the needs of ion optics or differential pumping system. Together with a pyrolysis
radical source, in-vacuum ion/radical reactions were performed and their associated
chemistry was studied. Radicals are important intermediates related to biochemical
processes and biological functions. There are very limited techniques to monitor the
reactive intermediates in-situ during a multi-step reaction in aqueous phase. On the other
hand, these intermediates can be “cooled down” and preserved into a single-step
procedure in gas-phase reactions since they only occur via collisions. Therefore, the
fundamental study of gas-phase radical ion chemistry will provide evidences of the
reactivity, energetics, and structural information of biological radicals, which has the
potential to solve puzzles of aging, disease biomarker identification, and enzymatic
activities.
Using the system described above, a new reaction between protonated alkyl amines
and pyrolysis formed cyclopropenylidene carbene was discovered, as the first
experimental evidence of the reactivity of cyclopropenylidene. Given the important role
of cyclopropenylidene in the combustion chemistry, organic synthesis, and interstellar
chemistry, it is highly desirable to establish a fundamental understanding of their physical
and chemical properties. The amine/cyclopropenylidene reactions were systematically
studied using both theoretical calculation and experimental evidences. A proton-bound
dimer reaction mechanism was proposed, with the amine and the carbene sharing a
proton to form a complex as the first step, which was closely related to the high gas-
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phase basicity of cyclopropenylidene. Subsequent unimolecular dissociation of the
complex yielded three possible reaction pathways, including proton-transfer to the
carbene, covalent product formation, and direct separation. These reactions were studied
with a variety of alkyl amines of different gas-phase basicities. For the covalent complex
formation, partial protonation on cyclopropenylidene within the dimer facilitates
subsequent nucleophilic attack to the carbene carbon by the amine nitrogen and leads to a
C-N bond formation. The highest yield of covalent complex was achieved with the gas-
phase basicity of the amine slightly lower but comparable to cyclopropenylidene. The
results demonstrated a new reaction pathway of cyclopropenylidene besides the
formation of cyclopropenium, which has long been considered as a “dead end” in
interstellar carbon chemistry.
Further reactivity study of cyclopropenylidene towards biomolecular ions was also
carried out for nucleobases, nucleosides, amino acids, peptides, proteins, and lipids. The
reaction to form proton-bound dimer for protonated biomolecular ions remained as the
dominant reaction pathway. Interestingly, other possible reaction pathways, such as
modifications of thiyl group or disulfide bonds, double bond addition, and single bond
insertion, were inhibited in gas-phase ion/carbene reactions. Such results inferred that the
reactivity of neutral species was not directly applicable to ion reactions, with the proton
involved in the gas-phase biomolecular ion reactions.
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CHAPTER 1. INTRODUCTION
1.1 Gas-phase biomolecular ion reactions and mass spectrometry instrumentations
Gas-phase ion reactions involve interactions between ions and various species
including molecules,1 electrons,2 ions,3 photons,4 reactive organic species,5 etc. Mass
spectrometry (MS), which analyzes charged particles by their mass-to-charge ratio (m/z),
is naturally involved with gas-phase ion chemistry and has been a powerful tool for
manipulating and analyzing gas-phase ions. Back in the 1950s’, researchers made great
efforts to decrease the pressure inside the vacuum chamber of mass spectrometers to
improve the resolution, accuracy and sensitivity. In the 1970s’, R. Graham Cooks at
Purdue University proposed and developed the early generations of tandem MS
instruments for complex mixture analysis using in-vacuum gas collisions.6 The most
common tandem MS technique is known as collision-induced dissociation (CID, a.k.a.
collision-activated dissociation, CAD), which utilizes collisions to break the selected ions
into fragments.7 These characteristic fragments, acting as fingerprints of the parent ion,
provide unambiguous structural identification of target analytes. The development of CID
promoted MS as a “gold standard” in chemical analysis in terms of both qualification and
quantification. The nature of CID is an energy-transfer ion/molecule reaction: the kinetic
energy of the ions accelerated by the electric/magnetic field through collisions between
the ions of interest and the inert buffer gas molecules, converts into internal energy,
further cleaving the weakest linkages of the ions to generate the fragments. The study of
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2
gas-phase reactions has been growing for both studies of basic chemistry and application
development in the past two decades. The advantage of gas-phase reactions, compared to
those in aqueous or liquid phase, is that the reaction occurs via direct collisions without
solvent clusters surrounded, where the energy barrier is lower, making it both kinetically
and thermodynamically favorable, e.g. proton-transfer reaction occurs more readily in
gas phase than in aqueous phase (Figure 1.1).8
Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase
Owing to the two revolutionary ionization techniques: electrospray ionization (ESI)9
and matrix assisted laser desorption/ionization (MALDI)10, which have been awarded the
Nobel Prize in Chemistry in 200211, intact ions of biomolecules such as peptides and
proteins can be produced from their condensed phase directly to the gas phase. MS was
then adopted for biomolecular identification and became the method of choice in
proteomics to study the structures and functions of proteins.12 The primary structure of a
peptide could be sequenced through the tandem MS methods.13 Two decades later, a
variety of dissociation methods have thrived in proteomics using different types of gas-
3
3
phase reactions.13 Table 1.1 summarizes the typical dissociation techniques, their
corresponding gas-phase reaction types, and the mass spectrometers that were involved in
protein sequencing. It demonstrates how gas-phase ion reactions, together with the
development of MS instrumentations, are closely linked to identifying biomolecules and
searching for disease biomarkers.6 Today, almost all of the proteomic strategies are based
on MS because it allows high throughput and structural analysis with high sensitivity.13-14
Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics
Tandem MS Gas-phase reaction type Instruments
Collision induced
dissociation (CID)
Ion/molecule
[M + nH]n+ + A → fragments
Triple
quadrupoles,
ion traps15
Electron capture
dissociation (ECD)
Ion/electron
[M + nH]n+ + e- → [M + H• + (n-1)H](n-1)+
→ fragments
Fourier transfer
ion-cyclotron-
resonance (FTI-
CR)16
Electron transfer
dissociation (ETD)
Ion/ion
[M + nH]n+ + A•- → [M + H• + (n-1)H](n-1)+
+ A → fragments
Ion traps17
Proton dissociation
(PD)
Ion/photon
[M + nH]n+ + hv → [(M + nH)n+]*→
fragments
Ion traps18
Metastable atom-
activated dissociation
(MAD)
Ion/metastable species
[M + nH]n+ + A* → [(M + nH)n+]* + A →
fragments
Ion traps19
Electron-detachment
dissociation
(EDD)
Ion/electron
[M - nH]n- + e- → [M -H• - (n-1)H](n+1)- →
fragments
Fourier transfer
ion cyclotron
resonance (FT-
ICR) 20
*Precursor peptide/protein ions shown in bold
Among all the gas-phase interactions in bioanalysis, reactions involving radicals
have been increasingly drawing attentions.5 Radical ions, which consist of at least one
unpaired electron are significantly more reactive than even-electron species, would
4
4
induce more gas-phase chemistry with two reactive functional groups within one species:
the charge and the radical sites. In living organisms, radicals are important intermediates
that are highly relevant to biochemical processes and biological functions. For instance,
radicals initiate modifications in proteins,21 DNAs,22 and lipids23 at the molecular level,
which are believed to be the cause of cell damage,24 cell death,25 aging,21a, 26
neurodegenerative diseases,25-26 and cancers27. Besides the damaging effects, radicals also
contribute to the catalytic activities of enzymes in several classes.28 Taking
ribonucleotide reductase as an example, a thiyl radical was used for catalysis,28b however,
the catalytic center is more than ten amino acid residues away from the radical site.28c
The puzzle of how the radical activity is controlled by the enzyme via such a long-
distance radical transfer process remains unsolved at the current stage. This is due to the
very limited techniques to monitor the reactive intermediates in-situ during a multi-step
reaction in the aqueous phase. On the other hand, these intermediates can be “cooled
down” and preserved into a single-step procedure since gas-phase reactions only occur
via collisions. Therefore, the fundamental study of gas-phase bio-radical reactions would
provide direct and/or indirect evidences of the reactivity, energetics, and structural
information of biological intermediates. The study might explain the intra- and inter-
molecular radical transfer in biological systems, and could potentially help to develop
drugs to cure serious diseases. Thus, developing a MS system suitable for gas-phase
reactions would be the very first step in achieving these goals. This review will start with
the concept of tandem mass spectrometry (MSn) and introduce gas-phase reactions at
different pressure stages of a tandem mass spectrometer: (1) the studies of gas-phase bio-
5
5
radical chemistry at atmospheric pressure and (2) MS instrumentations for gas-phase
reactions in vacuum.
1.1.1 Tandem mass spectrometry in gas-phase ion reactions
Tandem mass spectrometry (MSn) is a stepwise mass spectrometry analysis.7a A
direct MS analysis of ions produced from the ion source according to their m/z values is
called an MS full scan, also known as MS1. Selected precursor ions can undergo reactions
or fragmentations between two MS analysis stages, and the resulting product ions can be
analyzed in the next MS stage to generate MS2 spectra. Using a variety of mass analyzers,
MSn has been achieved with i) tandem-in-space mode, using sectors,29 triple
quadrupoles,30 and time-of-fight (TOF)31; or ii) tandem-in-time, using quadrupole ion
traps32 and Fourier transform ion cyclotron resonance (FT-ICR)33. Figure 1.2 gives a brief
summary of these MSn techniques.
Figure 1.2 Tandem mass spectrometry in space vs. in time
6
6
For the study of bio-radical (biomolecules that contain at least one unpaired electron)
chemistry, one additional reaction step must be applied to the biomolecules to create a
radical ion since soft ionization methods such as electrospray (ESI) and nano-
electrospray (nanoESI)34 usually generate lower-energy, even-electron species rather than
odd-electron radical ions. Bio-radical ions can be made directly at atmospheric pressure
through the interactions between the whole ion population and free radicals or
alternatively in vacuum through further reactions of the selected ionic species. Product
ions of ion/radical reactions at atmospheric pressure, for example in ESI plume, could
simply be introduced to the mass analyzer through atmospheric pressure interface (API)
and focusing lens. As a result, only MS1 is required to obtain a reaction spectrum.
Tandem mass spectrometry might be further applied to elucidate the molecular structure
of the product ions, as employed in almost all of the commercial MS systems. It normally
does not need modifications of the MS instruments; however, side reactions can occur
with multiple ionic reactants. Therefore, gas-phase ion reactions in vacuum with the pre-
selection of ions are preferred. Isolation of the precursor ions is performed before the
reaction and the reaction spectra are acquired in MS2. Yet, introducing a second flow of
neutral radicals or another ionic species in vacuum for reactions could be challenging and
usually requires extensive instrumentational modification. In the next two sections,
strategies for performing gas-phase ion reactions at atmospheric pressure and in vacuum
will be discussed.
7
7
1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure
The interaction of biomolecular ions and free radicals can be studied in the nanoESI
plume or in the spray bulk solution in atmosphere by adding a radical source such as a
low temperature plasma (LTP) generator35 (Figure 1.3a) and an ultraviolet (UV) lamp36
(Figure 1.3b) aside the ion source. The plasma or UV irradiation generates reactive
organic species (ROS) e.g. hydroxyl radicals (OHs), which subsequently react with the
ions and charged droplets in the nanoESI plume, or directly in the bulk solution. The high
number density and large cross-section at atmospheric pressure (760 Torr) facilitate the
reactions between two species. Rich chemistry can be observed through this simple
reaction approach for biomolecules such as disulfide-linked peptides and unsaturated
lipids. Note that although there is no restriction on MS instrumentation for these reactions
taking place before the product ions are introduced to the mass analyzer, MS systems
with versatile dissociation methods, e.g. beam-type CID or ion-trap slow-heating CID,37
could be essential to study the structure, reactivity, and further applications of the
reaction products.
Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low
temperature plasma (LTP)35 or ultraviolet (UV) lamp36.
8
8
1.1.2.1 Peptides
Disulfide linkage (S-S) is a type of posttranslational modification (PTM) formed by
the oxidation reaction between two cysteine residues among intra- or inter- peptide chains.
It is responsible for the stabilization of the native structures of proteins.38 The disulfide
bonds are considered difficult to break using CID due to its bonding energy being
significantly higher than those of the peptide backbones. Therefore, intra-molecular
disulfide bonds increase the difficulty in sequencing using even-electron based
dissociation methods.39 The conventional method for analyzing disulfide peptides is to
cleave the disulfide bonds using wet chemistry before sequencing (reducing disulfide
bond by alkylation39a). However, this causes the loss of constructional information.
Alternative methods are to use odd-electron radical-based dissociation methods (CID,40
ECD,16 ETD,17 EDD,20a, 20b UVPD,41 MAD,19 and etc.) in vacuum through additional gas-
phase reactions. Fortunately, disulfide bonds are very sensitive to free OHs, producing
different types of radical products (such as carbon-centered versus heteroatom-centered
radicals) with highly specific radical sites. As an example (Figure 1.4), the cleavage of a
disulfide bond occurs with the attack by hydroxyl radicals which yields three sets of
products: thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions.42
9
9
Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to
form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI plume: (a)
peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of AGC(TFTSC)K.42
Among the three radical species generated by the reaction in the nanoESI plume, thiyl
radicals have the highest reactivity while sulfinyl radicals are the most inert.42 Although
the cysteine sulfinyl radicals have low reactivity in aqueous solvents or under gas-phase
bimolecular reaction conditions, additional energy could trigger intramolecular reactions.
The energy applied by collisional activation can be sufficient to conquer the reaction
energy barrier but barely enough to cause backbone cleavage. Two reaction pathways of
radical migration were observed for a cysteine sulfinyl radical (SO•Cys) with either a free
thiol (-SH)43 or a disulfide bond (S-S)44 inside one peptide sulfinyl ion (Figure 1.5). The
discovered reaction channels indicate: (1) the disulfide bond scrambling could happen in
a protein based on the radical migration; (2) protein conformation and structural change
would be possible by the attack of the cysteine sulfinyl radicals; and (3) an oxidative
10
10
damage in terms of forming sulfinyl radicals, might potentially be cured with a nearby
free thiol group43.
Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free
thiol43 and (b) a disulfide bond44
Besides the reactivity study of bio-radical ions, ion/radical reactions in the ESI plume
enhance the backbone cleavage, thus improving the sequencing coverage.35 The
backbone cleavage for insulin, a protein with three disulfide bonds, occurred at an
efficiency rate of 26% obtained from even-electron based CID, but improved to about 60%
with odd-electron induced ECD or ETD methods.45 A favorable value of 75% backbone
cleavage has been achieved for cleaving disulfide bonds followed by applying a
conventional CID after the radical reaction in the ESI plume (Figure 1.6).35
11
11
Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with
a subsequent MS2 scan to improve sequence information of disulfide peptides5, 35
1.1.2.2 Lipids
Lipids are a group of biomolecules that function as the building blocks of cell
membranes, energy storage, and signaling transduction in living organisms.46 Individual
lipid47 or the lipid profile48 has been used as biomarkers for cancer diagnosis by ambient
ionization MS techniques, assuming that the lipid bio-synthesis pathways have been
changed in the tumor tissues.46 The degree of unsaturation in lipids (the number of C=C
bonds) and the location of double bonds determine the biomolecular structure and the
biological functions.49 Even-electron based dissociation methods are very helpful in
identifying the head group, the acyl chain composition, and the number of C=C bonds.50
However, the positions of C=C bonds, on the other hand, are difficult to be determined
since the bonding energy is much higher for C=C double bonds (~keV) than C-C single
bonds. This problem could not be solved with high energy collisions by sector or TOF-
12
12
TOF instruments because the C-C bonds are cleaved before the C=C bonds in a lipid. In
order to differentiate the unsaturated lipid isomers (different double positions in the acyl
chain), radical chemistry has been applied in vacuum or at atmospheric pressure. Radical-
directed dissociation (RDD) of lipid ions in vacuum yields fragmentation patterns
revealing information of double bond positions: a photo-caged radical precursor releases
the radical by irradiation, which forms a complex with the unsaturated lipids and
fragments are subsequently generated through CID.51 Alternatively, a Paternò–Büchi (PB)
reaction can be performed in the bulk solution of the nanoESI spray tip by UV lamp
irradiation (Figure 1.3b).52 A four-member ring is first formed through the interaction of
a ketone/aldehyde with an olefin, thus creating a set of low energy covalent bonds which
are then cleaved in the retro P-B reaction as shown in Figure 1.7.
Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin
together with possible retro P-B reactions.52
In the analysis of oleic acid (Figure 1.8),52 a covalent adduct m/z 339 of P-B reaction
of deprotonated oleic acid and acetone was formed with the 254 nm UV irradiation
(Figure 1.8a, b). MS2 CID of the product clearly showed three peaks in Figure 1.8c: m/z
281 corresponding to the retro P-B reaction to produce the original reactants
(deprotonated oleic acid); and one set of diagnostic ions with a mass difference of 26 Da
13
13
resulting from the fragments (structures 3 and 5) of two possible arrangements of P-B
reaction (isomers 1 and 2) (Figure 1.8d). By this means, lipid isomers (fatty acids and
phospholipids) in tissue samples with different double bond locations have been
identified and potential lipid biomarkers of diseases are under investigation.
Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS.
MS1 spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS2 CID of the
P-B reaction products at (c) m/z 339.3; and (d) structures of the diagnostics ions
(structures 3 and 5). The structures explain the origin of the 26-Da mass difference.52
1.1.3 Instrumentations for gas-phase ion reactions in vacuum
Compared to the reactions occurring at atmospheric pressure, gas-phase ion reactions
can also be carried out in vacuum. It provides one huge advantage of controlling
variables by having a single ionic reactant selected from the ion population, which largely
decreases the uncertainty of side reactions by unknown ions generated by the ion source.
14
14
The tradeoffs are however, the necessity of instrumentational modification and the
relatively low number density in vacuum (enough for reactions to occur) due to the low
pressures (usually 10-3 to 10-7 Torr). The first stage of tandem mass spectrometry (MS1)
can be used to isolate the precursor ions based on their m/z values. The subsequent gas-
phase reactions are then carried out in the following MS stages (MSn). Several types of
mass spectrometers have been modified to study gas-phase ion chemistry. For tandem-in-
space mass spectrometers such as triple-quadrupoles, penta-quadrupole, and selected-ion
flow-tube, a reaction chamber (designed for collisions) and at least one additional mass
analyzer for the following product analysis are required. On the other hand, the reactor
and mass analyzer can be integrated for trap-type instruments such as ion traps and
Fourier transform ion cyclotron resonance, which are able to store ions for a relatively
long reaction time and allow subsequent tandem MS. Hybrid instruments, involving a
trapping mode reaction chamber followed by a high accuracy mass analyzer for mass
analysis, are suitable for ion manipulation and reactions in the vessel, then providing a
high resolution product scan. Nevertheless, not all of these research prototype
instruments have been applied in gas-phase biomolecular ions reactions, especially for
bio-ion/radical reactions. This section will have an emphasis on the instrumentation: the
strategies to bring two species inside vacuum and the related reactions not limited to
biological ion interactions.
1.1.3.1 Sectors and time-of-flights (TOFs)
A sector mass spectrometer is a MS system using a static electric (E), a magnetic (B)
field, or a set of the combination of the two sectors to separate ions as a mass analyzer. In
15
15
the case of mass-analyzed ion-kinetic-energy spectrometry (MIKES),6a a magnetic sector
followed by an electronic sector was originally designed to study kinetic energy release
associated with metastable ion fragmentation by measuring the kinetic energy of mass-
selected ions. This instrument is most famously recognized as the first generation of
tandem mass spectrometer,6b, 53 with collision gas introduced between the two sectors
(Figure 1.9). The collisional energy in MIKES tandem MS could be as high as ca. keV,
which is sufficient to obtain fragments from most ionic species during tandem MS.
Guided ion beam (GIB) tandem MS is another sector instrument involved in gas-phase
chemistry. The ion beams selected by a magnetic sector are introduced to the reaction cell
(octapole ion guide) with reactant neutral of choice, followed by a double focusing
sector54 or a quadrupole55 as mass analyzer. This instrument has been widely used for the
determination of thermodynamic information for bimolecular reactions and CID.55
Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with
collision gas for chemical mixture analysis6a
As for a tandem time of flight56 (TOF-TOF, shown in Figure 1.10), the collisional
energy is able to reach keV level, similar to that of the sectors. Although TOF-TOF
16
16
instruments have been barely used in research of gas-phase ion reactions, TOF-TOF
provides a good dissociation methods coupled with MALDI for peptide identification.57
Furthermore, high mass resolution capability of TOF analyzer serves as an excellent
product mass analyzer for in-vacuum gas-phase reaction in hybrid instruments, which
will be further discussed in section 0.
Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56
1.1.3.2 Tandem quadrupoles
The triple-quadrupole (QqQ) mass spectrometer was developed in late 1970s30 and
remains as an outstanding MS2 instrument for chemical quantification. Triple-
quadrupoles have demonstrated tremendous capabilities in performing a set of MS2
experiments in tandem-in-space mode in three steps: (1) isolating precursor ions in Q1 by
applying certain amplitudes of radio-frequency (rf) and direct-current (dc) to allow ion
transmission at a narrow m/z window; (2) dissociative or reactive collisions in rf-only
collisional quadrupole q2 with activation energy ranging from 0 to tens of eV when using
17
17
inert or reactive buffer gas; and (3) product ion scan in Q3. The use of triple-quadrupoles
in studying ion/molecule reactions is valuable since the information of both precursors
and products are provided through different MS2 scan modes such as product ion scan,
precursor ion scan, neutral loss, and selected reaction monitoring (SRM) or multiple
reaction monitoring (MRM).58
Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59
The limitation inherent to triple-quadrupoles for ion/molecule reaction study is the
lack of structural analysis of the product ions with only two MS stages. Therefore, a
simple way to improve tandem-in-space instruments for ion/molecule reactions is to add
another MS stage, MS3. The penta-quadrupole (QqQqQ)60 (Figure 1.11) was designed
specifically for MS3 analysis for ion/molecule reactions. It employs two reaction rf-only
quadrupoles (q2 and q4) for reactions, and three rf/dc quadrupoles (Q1, Q3, and Q5) for
MS analysis. The tandem-in-space arrangement allows the study of ion/molecule
reactions involving mass-selected ions in two separate reaction regions (q2 and q4) by
using Q1, Q3, and Q5 for mass analysis. The first reaction region q2 can be set as
dissociative-collisional chamber for CID whereas q4 was filled with neutral reactants for
reactive collisions, or vice versa. A total of 15 different types of MS3 scan functions61 can
18
18
be achieved with 0-3 dimensions as shown in Figure 1.12. Each circle represents the
operation of one of the three mass analyzers, with a filled circle for a mass selection and
an open circle for MS scanning. Note that with all three mass analyzers transmitting fixed
masses, the consecutive reaction monitoring (three filled circles, 0 dimension) provides
mass spectra of one dimension. A remarkable 4D mass spectra were therefore acquired
using the entire MS3 data domain.62
Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3
dimensions. A fixed mass (filled circle) or variable masses (open circle) can be chosen for
reactions.61
An example of 3D familial scan is demonstrated in Figure 1.13.63 The ions produced
from 3-octanone by electron impact (EI) underwent CID in q2 via collisions with Argon
19
19
gas and the CID products of m/z 43 were selected for reacting with isoprene in q4; finally
the ion/molecule reaction product ions were analyzed by Q5. This experiment was useful
for ion chemistry since it provided reactivity information of isobaric and isomeric ionic
populations. Both sequential product spectra (horizontal) and sequential precursor spectra
(angles) can be extracted. Figure 1.13b shows the sequential precursors generating m/z 43
reacting predominantly by proton transfer that led to the product ions of m/z 81, whereas
Figure 1.13c identifies the sequential precursors producing acetyl cations of m/z 43,
which reacted with isoprene to form [4+2+] cycloadduct of m/z 111.
Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum
for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates. The angled
Q1 axis displays the precursors of m/z 43 ions, whereas the horizontal Q5 axis displays
the ion/molecule products of these CID-generated m/z 43 ions arising from reactions with
isoprene. Triple-stage sequential precursor spectra extracted from the 3D familial
spectrum by selection in Q5 of the final products of (b) m/z 81 and (c) m/z 111.63
20
20
Penta-quadrupole MS system has been proven to be suitable to study various
ion/molecule reactions.60 However, other types of gas-phase reactions such as ion/ion and
ion/radical reactions could barely be done on the penta-quadrupole mass spectrometer
without further modifications.
1.1.3.3 Flowing-afterglow selected-ion flow-tube (FA-SIFT)
Selected-ion flow-tube mass spectrometer has been used to study ion/molecule
reactions64 and also for quantitative analysis of trace, volatile organic compounds (VOCs)
using chemical ionization (typically proton-transfer reactions) with the selected precursor
ionic species along a drift tube within a well-controlled period. The applications extend to
gas analysis,65 breath testing,66 VOC monitoring from natural products67 etc., with pptv
level of detection limit. Together with the flowing-afterglow (FA) techniques, selected-
ion flow-tube has been employed to measure numerous ion-molecule kinetics data
especially those related to interstellar chemistry at a low temperature.68 In an FA-SIFT
instrument, the precursor ions generated by EI ionizer in the source flow tube (FA) are
sequentially selected by a SIFT quadrupole mass filter. The mass-selected ions flow
through the reaction tube where ion/molecule reactions occur with the neutral reactant
being introduced into the flow tube. The resulting product ions are scanned in the
detection quadrupole mass filter to produce the MS2 spectra. With the well-defined flow
time inside the reaction tube, the ion/molecule reaction kinetics can be acquired by
introducing neutrals at different positions along the drift tube to set a series of reaction
21
21
durations.68b FA-SIFT is also a tandem-in-space MS for ion/neutral reactions with the
reaction time in a well-defined fashion.
Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled
with a radical source for ion/radical reactions69
In 2004, this instrument was involved in the study of ion/radical reactions by adding
one additional radical source to the flow tube (Figure 1.14).69 The neutral radicals are
generated by a pulsed pyrolysis nozzle (also known as Chen nozzle70). Pulsed pyrolysis
has been an efficient means to produce gas phase radicals of high intensities in vacuum.71
It uses a silicon carbide tube for resistance-heating to dissociate organic precursors into
neutral radicals for the structural study of highly reactive species using infrared or
electronic spectra. Proton transfer reaction was observed between allyl radicals and
hydroniums, also associative detachment reaction between ortho-benzynes and
hydroxides, and radical recombination reaction between allyl radicals and benzyl radical
cations.69 The results firmly showed that FA-SIFT is capable of carrying out ion/radical
reactions with the additional radical source. No biological molecules have yet been
studied using this specific MS instrument.
22
22
1.1.3.4 Fourier transform ion cyclotron resonance (FTICR)
Different from tandem-in-space mass spectrometers, mass analyzers such as Fourier
transform ion cyclotron resonance (FTICR, Penning trap33) and quadrupole ion traps
(Paul trap72) are able to trap ions for a certain period of time, while carrying out multiple
steps of reactions and mass analysis. The advantage of tandem-in-time mode is that the
instruments do not require extra mass analyzers for performing additional stages of MS
analysis or reactions. The tradeoffs, compared to tandem-in-space, are longer duty cycles
and potential interference by the leftover neutral reactants.
FTICR employs a cyclotron cell that operates at a high vacuum of 10-7 to 10-8 Torr
for trapping. It consists of three pairs of adjacent electrodes assembled into a cubic,
cylindrical, or hyperbolic designs.73 A typical setup of FTICR is illustrated in Figure
1.15.74 The front and back electrodes, perpendicular to the strong magnetic field, function
as trapping plates to trap the ions inside the cyclotron cell. A radio frequency (rf) is
applied on the side plates to excite the trapped ions to induce the image current. The
detection plates (top and bottom) are used for monitoring the current induced by the ions
motions. A chirp is applied by sweeping the rf sweep from several kHz to several mHz to
excite the trapped ions. The induced image current is collected in the time domain. Mass
spectrum in frequency domain is then obtained with a fast Fourier transformation. The
mass resolution on FTICR is correlated to the strength of the magnetic field so that
accurate mass (< 1 ppm) can be obtained with large magnetic field (10T).
23
23
Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and
back), two opposite excitation plates (right and left), and two detection plates (top and
bottom). Ions enter the cell through the front trapping plate. Mass spectrum generated by
fast Fourier transformation.74
Similar to SIFT, FTICR was used in the study of interstellar chemistry at very low
temperatures68a and has been coupled with a pyrolysis nozzle.75 The reactions between
benzyl radical cations and pyrolysis-produced allyl radicals were studied, which formed a
carbon-carbon covalent bond. In another study, the protonated desR1-bradykinin ions
were mass-selectively isolated and underwent a hydrogen/deuterium (H/D) exchange
reaction with deuterium radicals generated by electron gun, which was one of the very
few reactions involving bio-ions and neutral radicals.76
Instead of carrying out reactions between bio-ions and neutral radicals, organic
radical ions can be generated in gas phase, trapped in FTICR, and then reacted with gas-
phase biomolecules. With laser-induced acoustic desorption (LIAD) techniques (Figure
1.16), neutral biomolecules, such as amino acids and peptide, can be made into gas phase
and interact with the trapped radical ions.77 Abstraction, addition, and proton-transfer
reactions have been observed for reactions between radical ions and biomolecules.
24
24
Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78
Besides ion/neutral (ion/molecule, ion/radical) reactions, FTICR is also well-used in
proteomic study, especially with the development of electron capture dissociation (ECD)
by McLafferty and coworkers in 1998.16, 79 To apply this technique, trapped positive
precursor bio-ions capture low energy electrons (<0.2 eV), forming odd-electron ions via
ion/electron reaction.80 Charge reduced ion [M+nH](n-1)+• is the main product of electron
capture, which causes peptide backbone cleavages.81 In contrast to CID, in which the
weakest bond is cleaved through an ergodic process, the major cleavage occurs at N-Cɑ
bond with ECD, resulting in c/z type of peptide ions.82 It is believed that an aminoketyl
radical is formed during the ECD, which subsequently produces c and z• fragments (or c•
and z) as shown in Figure 1.17.83 The reaction is fast, regarded as non-ergodic and a
perfect match with FTICR due to the low pressure in the cyclotron cell which allows
efficient electron capture.16
Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84
25
25
The c/z pair of ions is unique in radical peptide ion fragmentation compared to even-
electron species. One important feature of ECD is that the cleavages randomly occur,
without a preferred site except for the proline amino cleavage.85 Thus, almost all the N-
Cɑ bonds throughout the backbone of peptides could cleave to generate fragments, which
produces meaningful information for peptide and protein sequencing (Figure 1.18).86
Together with slow heating ion activation techniques in FTICR, such as sustained off-
resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton
dissociation (IRMPD), and blackbody infrared radiative dissociation (BIRD),87 more
internal energy is directed into the biological radical ions, and therefore more fragments
can be produced for proteins over 20 kDa. On the other hand, posttranslational
modifications can be preserved using ECD, which makes it possible to identify disulfide
linkage, glycosylation, phosphorylation and oxidation of proteins.2, 88
Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T
instrument.89
26
26
A variety of gas-phase reactions, including ion/molecule, ion/radical, and
ion/electron reactions, have been investigated using FTICR-MS to study the biological
molecules. FTICR-MS instruments with a large magnetic field are of high cost for
ownership and maintenance,45 in addition, the number density of neutral reactants is
relatively low due to the high vacuum requirements, which is not in favor of ion/molecule
or ion/radical reactions.
1.1.3.5 Quadrupole ion traps
Quadrupole ion traps are operated with radio-frequency (rf), which creates a
quadrupole electric field to trap the ions.72 Based on different geometries, quadrupole ion
traps can be categorized into 3D ion traps, and linear or 2D ion traps.72 The linear ion
traps (LITs) have increased trapping capacity. Quadrupole ion traps are effective tools for
in-vacuum gas-phase ion reactions because of their capability to store ions for long
reaction time (as high as 10 seconds) and the relatively higher pressure reaction
environment (10-3-10-5 Torr). Since gas-phase reactions occur via collisions, which is
highly dependent on the number density, the pressure inside quadrupole ion traps is three
orders of magnitude higher than FTICR, making them highly preferable for carrying out
gas-phase ion reactions. With these features, quadrupole ion traps, especially linear ion
traps, have been widely employed for studies of gas-phase ion reactions.
Neutral reagent vapor can be simply mixed with the collision gas for ion/molecule
reactions in the ion trap. An example is shown in Figure 1.19a. The neutral reactants were
leaked into Q3 linear ion trap of a modified QTrap 4000 (AB Sciex) mass spectrometer to
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investigate the reactivity of thiyl, sulfinyl, and perthiyl radicals.42 To introduce multiple
neutral reagents subsequently into ion trap mass spectrometers, a multi-valve interface
consisting of three neutral lines was designed,90 where three different ion/molecule
reactions could be carried out at the same time for fast neutral reagent screening (Figure
1.19b).
Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed
valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions.
As one of tandem-in-time mass spectrometers, ions are manipulated in one trap mass
analyzer to achieve multiple stages of mass analysis. For target analyte analysis, MSn
with CID can be easily carried out via isolating the precursor ions followed by multi-
collisional dissociation (ac activation induced rf heating). However, the structural
identification for the products of ion/molecule reactions can be tricky since neutral
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reactants remain inside the ion traps and can further react with the fragment ions. One
solution to this issue is to introduce two linear quadrupole ion traps in series as shown in
Figure 1.20,91 which borrows the idea of tandem-in-space MS systems yet keeps the
advantages of tandem-in-time MS systems. Ion/molecule reactions were carried out in the
first trap (LQIT1); the product ions of interest were then transferred into the second trap
(LQIT2) for structural determination; or vice versa, the CID products from LQIT1 could
be transferred to LQIT2 for ion/molecule reactions.
Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and
differential pumping. LQIT1 and LQIT2 are the first and second linear quadrupole ion
traps (LQIT).91
Gas-phase ion/ion reactions usually require longer reaction time and high abundance
for both positive and negative ions, and one of them needs to be multiply charged for the
reaction products to be analyzed by MS.92 Ion/ion reactions can be readily applied to
peptides and proteins since multiply charged ionic species can be produced through
spray-based ionization methods (ESI or nanoESI). Electron transfer dissociation (ETD),
proven to be one of the most useful applications of ion/ion interactions, occurs between
peptide or protein cations and radical anions in ion traps. Ion/ion reactions take place at
the millisecond level, much faster than ECD, which is limited by the slow electron
transferring rate. Thereby, ETD can be coupled with liquid chromatography.93 Reagents
such as anthracene and azobenzene are used to generate radical anions by chemical
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ionization (CI) or atmospheric pressure chemical ionization (APCI).94 These anions act as
electron donors due to their low electron affinity. Through electron transfer, the odd-
electron ions of reduced charge are produced and fragmented into c/y pairs from peptide
ions. A competitive proton-transfer reaction also occurs in ETD,95 giving even-electron
ions by losing a proton similar to the products in ECD through hydrogen loss. The
advantages of ETD include its low cost, the use of quadrupole ion traps instead of FTICR,
and the flexibility in ion manipulation. Low energy CID of all the resulting ions from
electron transfer reaction (supplemental collisional activation) or one specific charge-
reduced species (charge-reduced species CID) can be carried out with high sequencing
coverage.45, 96 Instrumentation of ion/ion reactions is generally more complicated,
compared to those for ion/molecule reactions, since two different ion beams are required.
Unlike 2D ion traps, 3D ion traps can store ions of both polarities.92, 97 As shown in
Figure 1.21, a home-built 3D ion trap instrument was constructed with three ion sources:
a positive and a negative ESI sources to generate multiply charged peptide/protein ions,
and an atmospheric sampling glow discharge ionization (ASGDI) source to produce ions
of either polarity for reactions.97-98 The turning dc quadrupole was used for selecting the
ion beam into the 3D ion trap through the holes on the endcap, while the ASGDI source
was directly pointing at the hole of the ring electrode. Studies of both proton-transfer and
electron transfer reactions were carried out using this configuration.
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Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two
electrospray ionization (ESI) sources and one atmospheric sampling glow discharge
ionization (ASGDI) source interfaced.98b
Linear (2D) ion traps have inherent advantages over 3D ion traps, such as higher
trapping capacity and higher trapping efficiency for externally injected ions, mutual
storage of ions of opposite polarities is not effective in conventional instruments92. Two
approaches have been reported for mutually trapping cations and anions: (1) applying rf
directly to at the end electrodes of the linear ion trap to create rf trapping along z-axis,17
so that positive and negative ions can be axially trapped simultaneously for reaction and
(2) using an unbalanced trapping rf to create a trapping field along the axial direction.99
Ion/ion reactions can also be obtained in transmission mode, whereby ions of one polarity
are trapped in the linear ion trap, and ions of the other polarity are injected through the
ion trap for reaction. An example is shown in Figure 1.22,100 the negative ions generated
by ASGDI were injected through Q3 and accumulated in Q2 before multiply charged
positive ions were injected through Q2, and the final products were analyzed in Q3. The
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radial injection of negative reactant ions into Q3and subsequent trapping in Q2 is not
efficient, which limits the effectiveness of the transmission mode. A pulsed dual ion
source consisting of +ESI/-ESI or ESI/APCI has been utilized to generate and introduce
ions of opposite polarities subsequently into Q2 linear ion trap for reaction through the
same ion pathway but different potentials on the optics.101 Proton-transfer and ETD
reactions have been investigated by employing the pulsed dual ion source.
Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an
atmospheric sampling glow discharge ionization source on the side of Q3 linear ion trap
for ion/ion reactions.100
Quadrupole ion traps are also feasible for studying ion reactions with metastable
species and photons based on specific modifications. Two modified instruments used in
these studies are shown in Figure 1.23. A pulsed fast atom bombardment was placed next
to a hole on the ring electrode of a 3D ion trap to generate metastable atoms to react with
peptide ions for inducing dissociation;19 laser emission was directed into a linear ion trap
via optics for peptide ion/photon reactions. Metastable atom-activated dissociation
(MAD),19 infrared multiple photon dissociation (IRMPD),102 and ultraviolet photon
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dissociation (UVPD)102a, 103 have been developed using the interaction of peptide ions
with metastable species, infrared, and ultraviolet photons, respectively, to produce odd-
electron ions for peptide sequencing. Ion/neutral radical reactions should also be
employed in quadrupole ion trap mass spectrometers if additional radical sources are
installed. The application of gas-phase ion/radical reactions in an ion trap mass
spectrometer is still under investigation.
Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB)
gun for metastable atom-activation dissociation19 and (b) a laser coupled linear ion trap
for infrared multiple photon dissociation (IRMPD)102b and ultraviolet photon dissociation
(UVPD)103.
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1.1.3.6 Hybrid instruments
Linear quadrupole ion traps have large trapping capacity and allow axial ion
injection, which makes them particularly attractive reactors for gas-phase ion reactions in
hybrid instruments. High accuracy mass analyzers (FTICR, TOF and Orbitrap) can be
coupled with linear ion traps to study a wide variety of gas-phase ion reactions. In this
section, two hybrid instruments, one with a TOF analyzer and one with an Orbitrap
analyzer are presented as examples of hybrid instruments for gas-phase ion reactions.
Quadrupole tandem time-of-flight (QTOF)
Figure 1.24 shows a quadruople tandem time-of-flight (QTOF) mass spectrometer
modified for ion/ion reactions.104 This instrument contained three quadrupoles (ion guide
Q0, mass filter Q1, collision cell Q2 as a linear ion trap) and an orthogonal reflectron
TOF analyzer for product analysis. A pair of pulsed ion source, ±ESI or nano-ESI
(+)/APCI (-), were placed in front of the MS inlet to produce the ionic reactants of
opposite polarities to study proton transfer reactions or ETD reactions. The reflectron
TOF significantly had improved mass resolving power and accuracy, compared to
quadrupole mass filter or Paul traps. The mass resolving power is about 8000 over a wide
mass range up to m/z 6,600 with a mass accuracy better than 20 ppm.
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Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer
for ion/ion reactions with dual ion source.104
Orbitrap Instruments
Orbitrap is a mass analyzer for exact mass measurement, developed by Makarov and
coworkers based on a King trap.105 The ions are trapped by the electrostatic field with a
centrifugal forces. Image current is measured for the harmonic axial motions. Hybrid
linear ion trap tandem Orbitrap (LTQ-Orbitrap) was commercialized in 2005.106 The
recent Orbitrap Elite (Figure 1.25)107 consists of two linear ion traps with different
pressures,108 a quadrupole mass filter, a C-trap, a collision cell and an Orbitrap mass
analyzer with an optional ETD reagent generator. The high-pressure ion trap is used to
cool down the ions whereas the low-pressure cell is for mass analysis or isolation. Mass-
selected precursor ions can be transferred along the quadrupole mass filter to the C-trap.
C-trap is used to cool and transfer ions into the Orbitrap. In the early design, C-trap was
also used to carry out slow-heating CID by increasing the rf, but with the restriction of
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low-mass cutoff (LMCO) through the rf ramping.109 Later on, another linear ion trap
(HCD collision cell) is used for high energy CID. Negative ETD reagent ions can be
introduced through ETD cassette into the HCD collisional cell as well. Gas-phase ion
reaction products are then transferred back to the C-trap before entering Orbitrap for
accurate mass measurement. Hybrid Orbitrap mass spectrometers have been used
intensively for the analysis of protein, glycan and biopharmaceutical applications.
Figure 1.25 Schematic of an Orbitrap Elite.107a
1.1.3.7 DAPI mass spectrometers
For the study of gas-phase ion reactions in hybrid instruments, more delicate
compartments can be installed to achieve multi-functions of high performance and high
compatibility, while compartments can also be reduced and simplified as long as the
basic requirements of in-vacuum ion reactions are satisfied. In all MS instruments
mentioned in the previous sections with atmospheric ion sources such as ESI and APCI,
the continuous atmospheric pressure interface (API) requires differential pumping system
to support the high vacuum for mass analysis as well as ion optical tools to guide the ions
into the analyzers. Discontinuous atmospheric pressure interface (DAPI),110 on the other
hand, is another solution to substitute for the massive interface (removing all ion optics)
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and differential pumping (largely reducing pumping requirements) in the conventional
MS instruments as shown in Figure 1.26. The original purpose of using DAPI together
with a rectilinear ion trap (RIT)111 was to miniaturize the mass spectrometers.112
Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with
differential pumping system and ion optics and (b) miniature DAPI-RIT mass
spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini 12 of Point-
of-care personal mass spectrometer.110, 113
The operation of a DAPI-MS consists of four steps (Figure 1.27a):110 (1) ions are
generated in the atmosphere by nanoESI or APCI before the DAPI opens; (2) the pinch
valve in DAPI opens for 10 to 30 milliseconds to transfer the ions into the trap in vacuum
chamber. In this process, rf is applied to trap ions and the pressure inside chamber
increases to 10-2-10-1 Torr; (3) after ion introduction, the DAPI closes and the ions stored
in the trap cool down for hundreds of milliseconds while the pressure decreases to 10-3
Torr; (4) at this pressure, mass-selective instability scan is used for mass analysis. One
scan cycle of a DAPI mass spectrometer usually takes one second, as the tradeoff for
breaking the barrier of pumping speed to not include a differential pumping system.
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Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b)
Manifold pressure measured during scanning, with an open time of 20 ms and a closed
time of 850 ms for DAPI.110
Multiple DAPIs can be implemented to introduce charged or neutral species for gas
phase ion reactions with minimum modification to an instrument. Note that the pressure
during the ion or neutral introduction is different from that for mass analysis (Figure
1.27b). This represents a unique opportunity for using a single trap to carry out gas-phase
reactions at a much elevated pressure, with reaction products later analyzed at 10-3 Torr
in a single scan cycle. One previous study114 by our research group has shown that two
beams of ions or neutrals can be introduced into a LIT sequentially or simultaneously
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through two DAPIs for gas phase ion processing, including gas flow CID, gas flow
assisted desolvation, and one ion/ion reaction. Simplified home-built mass spectrometer
with multiple DAPIs could be potentially constructed to investigate a variety of gas-phase
ion reactions, especially the interactions between biological ion and organic radicals, with
additional radical sources like the pyrolysis valve.71
1.2 Conclusion
Gas-phase reactions of biological ions especially those producing odd-electron bio-
radical ions, have been applied for biomolecule structural identification, disease diagnosis,
and enzymatic reactivity evaluation. Gas-phase bio-ion interactions can be carried out in
atmosphere or in vacuum. Significant basic chemistry and applications have been shown
in the study of gas-phase biological ion/radical reactions in the ESI plume or bulk
solution. Minimum instrumentational modification is required for such reactions at
atmospheric pressure, however no single ionic reactant can be selected prior to the
reactions. Ion manipulation and mass-selectively isolation can be achieved for gas-phase
ion reactions in vacuum, which on the other hand, require multi-stages tandem mass
spectrometry. Various tandem mass spectrometers of both tandem-in-space and tandem-
in-time configurations, have been demonstrated as useful tools for studying in-vacuum
ion/molecule, ion/ion, ion/radical, and ion/photon reactions. Among all the mass
analyzers, ion traps are feasible for all kinds of reaction types with long ion storage time.
Quadrupole linear ion traps are ideal reaction vessels for gas-phase reactions due to the
convenience of introducing ionic or other reactants and the relatively high pressure for
reactions. Hybrid instruments employ linear ion traps as the reactor and accurate mass
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analyzers for product analysis, which represents an effective way of improving the
instrumentational performance in studying gas-phase reactions. On the other hand, a
multi-DAPI mass spectrometer presents an opposite approach that is “just good enough”
for the study, with the simplest means to introduce reactants in a controlled environment.
This thesis features a DAPI-RIT-DAPI mass spectrometer that has been developed
with a rectilinear ion trap as both the reactor and the mass analyzer, and two pulsed
interfaces to introduce ions and other species for reactions. Ion/molecule and ion/ion
reactions were used to characterize the established system at an elevated reaction pressure
and with the energetic gas flow by the DAPIs. With an additional radical source, a novel
ion/radical reaction between protonated alkyl amines and pyrolysis-generated C3H2
carbene bi-radicals to form a C-N covalent bond was discovered with the
instrumentational setup shown in Figure 1.28. This reaction was further applied to
biological ions such as nucleobases, nucleosides, amino acids, peptides, and proteins.
Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis
radical source for the study of gas-phase ion/radical reactions.
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CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME-
BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE
ION/MOLECULE AND ION/ION REACTIONS
2.1 Introduction
Mass spectrometry (MS) has been widely used to study gas-phase ion chemistry via
inducing interactions or reactions between ions and matter in vacuo, such as neutrals,1
radicals,2 electrons,1c, 3 ions,4 photons,5 surfaces,6 metastable species,7 etc. Besides being
used as a mass analyzer, a quadruple ion trap can serve as a reactor for studying gas-
phase reactions with capabilities of manipulating the trapped ions in a controlled-time
fashion at relatively high pressure for reactions. Owing to the development of MS
systems with atmospheric pressure ionization sources such as electrospray ionization
(ESI)8 and atmospheric pressure chemical ionization (APCI)9, ions of intact molecules
can be generated directly from condensed phase at atmospheric pressure. The
atmospheric pressure interface (API) is an essential component in these mass
spectrometers, through which the ions are transferred from atmosphere to the mass
analyzer in the vacuum.
The APIs adopted in commercial mass spectrometers typically perform at
continuous ionization and ion introduction mode. The continuous APIs require a
differential pumping system with a large pumping capacity to support multiple vacuum
regions at different pressures. Delicate ion optics is used to transfer the ions through
different regions.10 Instrumentational modification is always needed to conduct gas-
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phase reactions on a mass spectrometer consisting of a single API. For instance, neutral
reactant needs to be leaked into the vacuum for ion/molecule reactions,11 which is
practically done for quadrupole ion trap or filter instruments through the collision gas
introduction line. To implement ion/ion reactions, a different ionization interface is
typically required to introduce an ion beam of the other charge polarity,12 which can
demand significant efforts in the instrument modification. An alternative solution is to
use a discontinuous atmospheric pressure interface (DAPI) for introduction of ions or
neutrals, which was initially developed for miniature mass spectrometers and has been
demonstrated to be effective with small pumping systems.13 In a DAPI, a pinch valve is
used to control the opening of the interface (10 to 30 ms) and the ions are transferred
from the atmosphere directly to the ion trap mass analyzer in the vacuum. During the ion
introduction period, the pressure inside the vacuum manifold increases to 10-2 to 10-1 Torr.
The ions are trapped at the elevated pressure and mass analyzed after a delay of several
hundred milliseconds to allow the pressure dropping back to 10-3 Torr.13b DAPI can be
implemented even with bent capillaries without losing ion intensity significantly,14 which
provides flexibility for the instrument modification. It has been demonstrated in a
previous study15 that multiple beams of ions or neutrals can be introduced into a
rectilinear ion trap (RIT)16 sequentially or simultaneously through two DAPIs for gas
phase processes including collisional induced dissociation, gas flow assisted desolvation,
and ion/molecule or ion/ion reactions.
In this study, we adopted the simple DAPI-RIT-DAPI configuration previously
proposed15 to set up an instrument that could potentially be used for gas-phase ion
reactions with multiple beams of ions or neutrals conveniently introduced through the
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two DAPIs. A distinct feature of the MS systems with DAPI is that the in-vacuum
pressure varies during a scan cycle and the ions trapped in vacuum experience stronger
and more energetic gas flows. This represents a unique opportunity for using a single trap
to carry out gas-phase reactions at a condition different from MS analysis of the reaction
products in a single scan cycle. The pressure changes and gas flow impact can be critical
to gas-phase reactions and need to be characterized for studying complicated reactions.
Herein, the gas dynamic effects on ion internal energy and ion reactivity as well as the
pressure effect on the reaction rate have been investigated. Two types of ion reactions
were used to test this system, including ion/molecule and ion/ion reactions. Ion/ion
reactions have been widely applied in proteomics1c and they have been made available on
several types of commercial instruments. We are interested to test the performance of our
instrument with extremely simplified ion optics for ion/ion reaction applications.
Ion/molecule reactions, on the other hand, offer a great means for studying gas-phase ion
structures and energetics. Many research labs have modified their instruments to facilitate
this type of fundamental studies. In this work, we chose ion/molecule reactions involving
peptide radical ions because the structures and reactivity of radical ions are very sensitive
to instrument conditions, e.g. energy deposition associated with gas collisions during the
ion transfer and storage.17
2.2 Instrumentation
The home-built DAPI-RIT-DAPI mass spectrometer was developed using the
vacuum chamber with inside dimensions of 35 × 25 × 25 cm. The manifold was pumped
by a 210 L/s turbo pump (THH 262 P, Pfeiffer Vacuum Inc, New Hampshire) and a 30
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m3/h rotary vane pump (UNO-030M, Pfeiffer Vacuum Inc., New Hampshire). A nanoESI
emitter consisting of a tip-pulled (i.d. < 20 m) boron silica glass capillary (0.85 mm i.d.
and 1.5 mm o.d.) was used to generate ions in front of DAPI-1 (the left DAPI in Figure
2.1a). Other reagents were introduced by the gas flow through DAPI-2. A pinch valve
(ASCO 390NC24330, ASCO Scientific, Florham Park, NJ) with a semi-conductive
silicone tubing (3 cm long, ∼350 Ω resistance, with 1.6 mm i.d. and 3.2 mm o.d.) was used
for each DAPI. Capillary 1 (10 cm long and 0.5 mm i.d.) was attached to DAPI-1;
Capillary 2 (25 cm long and 0.75 mm i.d.) and Capillary 3 (5 cm long with various i.d.s
tested) were attached to DAPI-2. All the capillaries were grounded. The DAPI-1 and 2
were controlled separately each by a 0-24 V pulsed dc signal from the control electronics.
An RIT with dimensions x0 = 5 mm, y0 = 4 mm, and z = 40 mm was operated with a
single-phase radiofrequency (rf) at 850 kHz for ion storage and MS analysis. The end
electrodes were made from electrogalvanized steel mesh electrodes (0.009” Wire,
McMaster-Carr, Aurora, OH) approximately 2 mm away from the capillaries of each
DAPI to allow ion/neutral injection. A previous study with simulations18 has shown that a
mesh electrode, in comparison with a plate electrode with a center hole, can minimize the
disturbance to the trapped ions by the gas flow from the DAPI while allowing high
throughput of the ions. A set of control electronics, from a Griffin Minotaur 300 (FLIR
Systems, Inc., Wilsonville, OR), was used for the instrument control. A 30 V dc was
applied on both end electrodes to create a dc trapping potential well along the z direction.
Resonance ejection was implemented for MS scan with a dipolar ac signal at 300 kHz (q
= 0.81). The conversion dynode of the detector was set to 0 V for positive ion mode and
+4000 V for negative ion mode. The isolation of the trapped ions was achieved by a
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notched SWIFT (stored waveform inverse Fourier transform). Collision-induced
dissociation (CID) was implemented by dipolar excitation with a single-frequency ac
applied between the x electrodes of the RIT. The typical nanoESI spray voltage was set to
+/-1600 V in positive/negative ionization mode. Multiple triggers were provided from the
control electronics for synchronizing the operations of nanoESI, DAPIs, and MS analysis.
All the spectra presented are the average of three continuous scans.
2.3 Materials
S-nitrosoglutathione (γ-E(NOC)G), dimethyl disulfide, allyl iodide, angiotensin I
(single letter sequence: DRVYIHPFHL), pentachlorophenol (PCP), 9-anthracene
carboxylic acid, cocaine, and benzylpyridinium thermometer ions were purchased from
Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different concentrations (5-100
μM) of the samples were prepared with methanol/water (1:1, v/v) with 0.2 % acetic acid
added to increase the intensity of protonated species if necessary.
2.4 Results and discussion
2.4.1 System configuration
The DAPI-RIT-DAPI system (Figure 2.1a) consists of two DAPIs, one RIT, and one
detector. The general scan function for gas-phase reactions shown in Figure 2.1b was set
as following. DAPI-1 was opened for 10-30 ms to introduce ions into the RIT; the ions of
interest for reaction were isolated using SWIFT, and CID was performed and followed by
another isolation if a fragment ion was used for reactions; the second DAPI, DAPI-2 was
opened to introduce other reagents into the RIT, which can be neutral molecules for
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ion/molecule reactions or ions for ion/ion reactions. Capillary 3 could be easily switched
with capillaries of different inner diameters from 0.125 to 0.75 mm, which was selected
based on the desirable pressure or reaction time (for neutrals) for the reaction. An
opening time as long as 250 ms could be applied with a 5 cm capillary of id 0.125 mm
and multiple introductions could also be implemented.15 The species introduced from
both DAPIs interact with each other inside the RIT during the cooling period after DAPI-
2 was closed. The final product ions were analyzed by rf scan with resonance ejection at
q = 0.81. CID could also be performed to the product ions.
Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General
waveforms of the in-trap gas-phase reactions
Two types of reactions, ion/molecule and ion/ion reactions, were performed using
the DAPI-RIT-DAPI system to show the feasibility of this configuration. The product
spectra are shown in Figure 2.2 and Figure 2.3a, respectively. For ion/molecule reactions,
peptide thiyl radical ions [γ-E(S•C)G+H]+ (m/z 307) were generated by CID19 of the
protonated S-nitrosoglutathione [γ-E(NOC)G+H]+ (m/z 337) introduced through DAPI-1
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(Scheme 2.1) and reacted with the allyl iodide vapor introduced through DAPI-2
(capillary 3: i.d. 0.25 mm, opening time 25 ms). The reaction time was set to 750 ms after
closing DAPI-2 and before rf scan. The thiyl radical located on the cysteine side-chain
attacked the C-I bond within allyl iodide, leading to the formation of allyl adduct at m/z
348 (+•C3H5, +41 Da) and iodine adduct at m/z 434 (+•I, +127 Da). The reactions
between the thiyl radical with dimethyl disulfide was also observed with product ions at
m/z 354 (+•SCH3, +47 Da). The reaction phenomenon is consistent with literature reports
on peptide thiyl radical species (Scheme 2.2).20 The details of the thiyl radical reaction
will be further discussed later to characterize the gas flow effect on the radical reactivity.
Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide
Scheme 2.1
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Scheme 2.2
For ion/ion reactions, protonated angiotensin I (DRVYIHPFHL) generated via
positive mode nanoESI (77 μM) was introduced into the RIT through DAPI-1 with a
pulsed open time of 30 ms (Figure 2.3b). The triply charged ions (m/z 433) were isolated
at q = 0.80 after 250 ms of cooling time (Figure 2.3a inset). The anion reagent was
generated with negative mode nanoESI of 37 μM pentachlorophenol (PCP, MW: 266.34)
and introduced through DAPI-2 with capillary 3 of 0.25 mm i.d. for 60 ms. The single-
phase rf applied on the RIT allowed efficient trapping of both cations and anions with the
dc voltage on the end electrodes set to zero. A low-mass cutoff at m/z 220 and a reaction
time of 260 ms were used (scan function shown in Figure 2.3d). As shown in Figure 2.3a,
product ions resulted from proton transfer reactions, i.e., doubly charged angiotensin I at
m/z 649, can be well detected. Noticing that fragment ions such as b4+ and y4
+ 21 were
observed, which was not common in ion/ion reactions. This phenomenon is probably due
to an impact of the second gas flow (collisional activation) on the triply charged peptide
ions. Same fragments were also observed when using the same scan function (DAPI-2
opened), however, with the nanoESI source turned off for anion production (Figure 2.3c).
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Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with
deprotonated pentachlorophenol anions (insert: Isolation of the triply charged species); (b)
MS spectrum of Angiotensin I without opening DAPI-2; (c) MS spectrum of isolated
triply charged peptide ions with DAPI-2 opening but the nanoESI source being turned off
for anion production; and (d) aveforms of the ion/ion reaction (dc was set to -10 V during
mass analysis).
Based on the results of Figure 2.2 and Figure 2.3, both ion/molecule and ion/ion
reactions could be performed on the DAPI-RIT-DAPI system, while the pressure change
and the energy deposition by the gas flow occur during the DAPI opening. As discussed
above, these two features could play important roles in gas-phase reactions. In the
following studies, we focused on the characterization of these two factors for carrying on
the gas-phase ion reactions using the DAPI-RIT-DAPI system.
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59
2.4.2 Pressure effect on gas-phase ion reactions
With the DAPI MS systems, the pressure in the ion trap can rise to 10-1 Torr or
higher after the opening of DAPIs, which is much higher than the pressure for
conventional MS analysis using quadrupole ion traps (mTorr or lower). After the DAPI is
closed, the pressure is gradually pumped down. By varying the delay time for performing
the CID, the dissociation reactions can be carried at different pressures. As shown in a
series of CID experiments (q = 0.30) of cocaine ([M+H]+, m/z 304, 3 μM, Scheme 2.3) at
different trap pressures, the optimal fragmentation efficiency (defined as the fraction of
fragment ion intensity over total ion intensity) occurs at ~8 x 10-4 Torr as shown in Figure
2.4. The activation time was set to 30 ms and the amplitude of the excitation AC was 200
mV. The relatively lower fragmentation efficiency at pressures lower than 5 x 10-4 Torr is
likely due to the lower collision rate at lower pressures. The decrease in fragmentation
efficiencies at pressures higher than 3 mTorr could be due to the significantly enhanced
damping effect that cools the ions to the center of the trap.22
Scheme 2.3
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Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different
pressures. Activation time 30 ms, amplitude 200 mV.
CID was also performed (q = 0.30) for deprotonated 9-anthracene carboxylic acid
(m/z 221, 5 μM) at different pressures (Figure 2.5). When the CID was performed at
about 6 mTorr (200 ms after DAPI-1 opening), only product ion m/z 195 was observed
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(Figure 2.5a); At pressure of about 2 mTorr (500 ms delay), another product ion m/z 177
was observed in addition to m/z 195 (Figure 2.5b); Product ion m/z 177 became the only
product when CID was carried out at 1 mTorr or lower (780 ms delay) (Figure 2.5c). The
ions m/z 177 could be obtained also by isolating and activating m/z 195 (Figure 2.5d).
The CID activation time was 30 ms. In-trap CID experiments were performed on a
commercial triple quadrupole/linear ion trap (QTRAP 4000, AB Sciex, Toronto, Canada)
for product verification. The precursor ions at m/z 221 were isolated in Q1 mass filter and
fragmented in Q3 linear ion trap via on-resonance CID. As shown in Figure 2.5e, the only
fragment pathway observed was CO2 loss, giving rise to the anthracene anion at m/z 177,
which has been demonstrated as an ETD reagent.23 This result only matches the CID
results obtained at low pressure when using the DAPI instrument (Figure 2.5c). Given the
high reactivity of anthracene radical anions, m/z 195 observed under higher pressures
(Figure 2.5a and b) might result from sequential ion/molecule reactions of m/z 177 with
trace H2O in the trap. To test this hypothesis, water vapor was mixed into the collision
gas on QTRAP 4000. The anthracene anions (m/z 177) were stored in Q3 linear ion trap
for various reaction times with H2O at the trap pressure of 4 × 10-5 Torr. Figure 2.5f
shows the data after 3 s reaction time, in which m/z 195 can be clearly detected. A peak at
m/z 221 was also observed, which is probably due to reaction of the anthracene anions
with trace CO2 in the water vapor. Note that the reaction time on QTRAP 4000 was long
(3000 ms), yet anthracene anions at m/z 177 remained as the base peak, while 100% yield
of water adduct was observed with the DAPI instrument at only 600 ms reaction time
(Figure 2.5a).
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Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6
mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay, from the
DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and blue columns illustrate
the positions of ion introduction, dipolar ac excitation, and MS scan, respectively; (d)
Ion-trap CID spectrum of m/z 195 on DAPI-RIT-DAPI system; (e) Ion-trap CID
spectrum of deprotonated 9-anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule
reactions of deprotonated anthracene ions with water vapor on QTRAP 4000. The CID
activation time was set to 30 ms on DAPI-RIT-DAPI system.
As a summary, an optimal CID efficiency was obtained at about 0.8 mTorr and
ion/molecule reactions happened at much faster rates at higher pressures (6 mTorr) using
the DAPI-RIT-DAPI instrument and therefore the reaction products can be obtained with
short reaction time.
2.4.3 Dynamic gas flow effect on gas-phase ion reactions
According to the previous simulation study,18 the gas expansion after a DAPI is
significantly larger and the gas velocity is much higher than those for a continuous API.
To characterize the internal energy distributions of the ions via DAPI introduction, the
“survival yield” method, which has been developed to determine ion internal energies,24
was applied in our study. A comparison was made between the DAPI using the home-
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built instrument and a continuous API using the QTRAP 4000. A methanol/water (1:1,
v/v) solution containing the following five compounds at 10-4 M each, p-
chlorobenzylpyridinium chloride (p-Cl), p-methylbenzylpyridinium bromide (p-CH3), p-
methoxybenzylpyridinium tetrafluoroborate (p-OCH3), p-nitrobenzylpyridinium bromide
(p-NO2) and p-cyanobenzylpyridinium chloride (p-CN), was used to generate the
thermometer ions by nanoESI. The ions were introduced into the RIT directly through
DAPI-1 (DAPI opening time of 15 ms with a low-mass cutoff m/z 60) in the home-built
instrument, or through the continuous API into Q3 linear ion trap of QTRAP 4000 with
declustering potential of 10 V, collisional energy of 5 V, and Q3 entry barrier of 2 V (the
minimum energy setup on the commercial instrument), respectively. Figure 2.6a shows
the breakdown curves that were calculated and plotted using the methods described
previously.24b In detail, the critical dissociation energies (E) of the five benzylpyridinium
ions (p-OCH3, p-CH3, p-Cl, p-CN, and p-NO2) are 1.51, 1.77, 1.90, 2.10, and 2.35 eV,
respectively. The survival yield (SY) was calculated as I(parent ion)/[ I(parent ion) + Σ
I(fragment ion)], where I(parent ion) and I(fragment ion) represent the intensities of
precursor and fragment ions, respectively. The corresponding internal energy distribution
P(E) (Figure 2.6b) was then calculated by taking the derivative of the breakdown curve in
Figure 2.6a. The internal energy distribution associated with DAPI is about 0.4 eV higher
than the continuous API.
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Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b)
Internal energy distributions of DAPI vs. continuous API for ion introduction;
Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms with
capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the fragment of p-R (R =
OCH3, CH3, Cl, CN, NO2).
To conduct reactions on the DAPI-RIT-DAPI instrument, a second reagent species
was introduced with a gas flow from DAPI-2 after the ions injected through DAPI-1 had
been trapped and cooled. It was observed that the second gas flow could cause the
dissociation of the ions of small organic compounds from our previous studies15 and
triply charged peptide ions. Therefore, the ion internal energy change due to the second
gas flow was investigated using the thermometer ions. After the thermometer ions were
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introduced through DAPI-1, their corresponding molecular ions were isolated and the
DAPI-2 was opened while an rf voltage corresponding to a low-mass cutoff of m/z 60
was used. No fragmentation of the thermometer ions was observed with capillary 3 of
0.125 mm or 0.25 mm i.d., when DAPI-2 was open for up to 250 ms. The most fragile p-
OCH3 ions fragmented with a capillary 3 of 0.5 mm i.d. and an opening time of 20 ms
(Figure 2.6c), while p-OCH3, p-CH3, and p-Cl started to fragment with capillary 3 of 0.75
mm i.d. DAPI-2 opening time 20 ms (Figure 2.6d). The disturbance and the energy
deposition to the trapped ions could be controlled by selecting capillary 3 of proper
conductance, i.e. capillary i.d. smaller than 0.5 mm. However, for introduction of ions for
a reaction, the transfer efficiency decreases significantly when the capillary i.d. is smaller
than 0.25 mm.13b These results indicate that the second gas flow led to an energy
deposition to the trapped ions. Its influence on the reactivity of the radical ions was
further studied with the reactions between glutathione thiyl radical ions and dimethyl
disulfide.
As discussed earlier, glutathione thiyl radical ions could be produced from
protonated S-nitrosoglutathione (30 μM) through CID by a facile NO loss (Scheme 1).19,
20b Figure 2.7a shows a MS spectrum with a DAPI-1 opening time of 15 ms using a scan
without applying excitation AC for CID. A fragment ion at m/z 307 was observed,
corresponding to the loss of a NO from the precursor (m/z 337). This ion presumably is
due to additional internal energy deposition to the precursor during ion introduction
through DAPI-1, for which the average internal energy distribution is 0.4 eV higher than
that from continuous API as shown in Figure 2.7b. For the ion/molecule reaction, the
protonated S-nitrosoglutathione (m/z 337) was isolated and excited to produce thiyl
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radical ions (m/z 307) through CID (Figure 2.7b) for subsequent ion/molecule reactions.
The head-space vapor of dimethyl disulfide (placed in a glass tube with a plastic
Swagelok connected to capillary 3) was introduced into the vacuum chamber for reaction
through DAPI-2. Dissociative addition of the dimethyl disulfide to thiyl radical was
observed to occur (Figure 2.7c), leading to the formation of glutathione methyl disulfide
(Scheme 2) at m/z 354. The reaction yield, defined as the fraction of the product intensity
(m/z 354) over the total ion intensity (sum of m/z 307 and m/z 354), was 70% under this
condition.
Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a)
MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by CID of
protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals and dimethyl
disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25 ms; q=0.35 for thiyl
radical ions); (d) Ion/molecule reactions between isolated m/z 307 from panel (c) with
dimethyl disulfide.
Scheme 2.2
HOOCNH
OS
HN
NH2 O
COOH + H
+
m/z 354+ ●SCH3 (47 Da)
m/z 307
CH3SSCH3
●
HOOCNH
OS
HN
NH2 O
COOH
S
+ H
+
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Multiple introductions of the reactant species within a single scan through DAPI-2
could be implemented using the method described previously.13d Higher reaction yield
should be expected with longer DAPI opening time and/or higher number of neutral
introduced; however, no significant difference in the reaction yield was observed for the
different scan functions with different DAPI-2 opening times and various numbers of
injections (Figure 2.8).
Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times.
A new scan function was designed and implemented to isolate the thiyl radical ions
m/z 307 (Figure 2.7c) after one reaction cycle and then allowed to react with the dimethyl
disulfide introduced again from DAPI-2 (capillary 3 i.d. 0.125 mm; DAPI-2 opening time
25 ms). Surprisingly, no product ions were observed (Figure 2.7d). This indicates that a
portion of thiyl radical ions had been converted into nonreactive species, presumably Cα-
radical ions,17, 19 during the first reaction cycle. In fact, the energy barrier of thiyl radical
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isomerization is relatively low (7.4 kcal/mol).17 It has been demonstrated that in-source
fragmentation or in-trap CID can promote this process and lead to the loss of thiyl radical
reactivity. In order to confirm this hypothesis, an ac excitation was used to activate the
thiyl radicals immediately after they were produced and isolated; dimethyl disulfide was
then introduced for reactions. The reaction yield decreased as the ac amplitude for
excitation increased (Figure 2.9). Specifically, the reaction yield was only 15% with an ac
excitation voltage of 0.07 V and the fragments of the thiyl radical ions, m/z 289 due to
loss of water and m/z 232 ([b2-H]+•) were also observed (Figure 2.9c).19
Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V;
(b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions.
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These results suggest that more stable Cα-radical ions could be formed with energy
deposition through an excitation, either by ac activation or gas flow. The side reactions
might occur due to the gas flow with DAPI opening.
2.5 Conclusion
Use of simple instrument configuration of multiple DAPIs for gas-phase ion
chemistry study was explored with a home built DAPI-RIT-DAPI system. The
discontinuous atmospheric pressure interfaces enable introduction of multiple reactants
with simple instrument configuration. The in-trap pressure varies during a scan, which
also allows a reaction to be carried out at a high pressure while MS analysis of products
at a much lower one. The gas dynamic effect on the internal energy deposition to the ions
has been studied with thermometer ions. The ion/molecule reaction between glutathione
thiyl radical and dimethyl disulfide was investigated to demonstrate the feasibilities of the
DAPI-RIT-DAPI system, where a radical immigration reaction occurred at the same time
of the dissociative addition reaction. With the simplicity of the configuration and the
easiness in instrumentation modification, a variety of sources for ions and radicals, such
as synchronized discharge ionization,25 low temperature plasma ionization,26 UV light
irradiation,27 and pyrolysis nozzle,28 can also be coupled with this system in the future for
investigation of the gas-phase reactions.
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16. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G.,
Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass
analyzer. Anal. Chem. 2004, 76 (16), 4595-4605.
17. Rauk, A.; Armstrong, D. A.; Berges, J., Glutathione radical: Intramolecular H
abstraction by the thiyl radical. Can. J. Chem. 2001, 79 (4), 405-417.
18. Garimella, S.; Xu, W.; Ouyang, Z., Simulation of Transient Rarefied Gas
Expansion for Discontinuous Atmospheric Pressure Interface (DAPI) Using Direct
Simulation Monte Carlo (DSMC). American Society for Mass Spectrometry 60th Annual
Conference Proceedings: Ion Manipulation, Analysis and Detection: New Developments
2012, ThOF (PM), 349.
19. Zhao, J.; Siu, K. W. M.; Hopkinson, A. C., Glutathione radical cation in the gas
phase; generation, structure and fragmentation. Org. Biomol. Chem. 2011, 9 (21), 7384-
7392.
20. (a) Osburn, S.; Berden, G.; Oomens, J.; O'Hair, R. A. J.; Ryzhov, V., Structure
and Reactivity of the N-Acetyl-Cysteine Radical Cation and Anion: Does Radical
Migration Occur? J. Am. Soc. Mass Spectrom. 2011, 22 (10), 1794-1803; (b) Tan, L.; Xia,
Y., Gas-Phase Reactivity of Peptide Thiyl (RS center dot),Perthiyl (RSS center dot), and
Sulfinyl (RSO center dot) Radical Ions Formed from Atmospheric Pressure Ion/Radical
Reactions. J. Am. Soc. Mass Spectrom. 2013, 24 (4), 534-542.
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21. (a) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F., Anion
dependence in the partitioning between proton and electron transfer in ion/ion reactions.
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Fragmentation Reactions of Multiply-Protonated Peptides and Implications for
Sequencing by Tandem Mass-Spectrometry with Low-Energy Collision-Induced
Dissociation. Anal. Chem. 1993, 65 (20), 2824-2834.
22. Tolmachev, A. V.; Vilkov, A. N.; Bogdanov, B.; Pasa-Tolic, L.; Masselon, C. D.;
Smith, R. D., Collisional activation of ions in RF ion traps and ion guides: The effective
ion temperature treatment. J. Am. Soc. Mass Spectrom. 2004, 15 (11), 1616-1628.
23. Huang, T. Y.; Emory, J. F.; O'Hair, R. A. J.; McLuckey, S. A., Electron-transfer
reagent anion formation via electrospray ionization and collision-induced dissociation.
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24. (a) Gabelica, V.; De Pauw, E., Internal energy and fragmentation of ions
produced in electrospray sources. Mass Spectrom. Rev. 2005, 24 (4), 566-587; (b) Nefliu,
M.; Smith, J. N.; Venter, A.; Cooks, R. G., Internal energy distributions in desorption
electrospray ionization (DESI). J. Am. Soc. Mass Spectrom. 2008, 19 (3), 420-427; (c)
Wang, H.; Liu, J. J.; Cooks, R. G.; Ouyang, Z., Paper Spray for Direct Analysis of
Complex Mixtures Using Mass Spectrometry. Angew. Chem.-Int. Edit. 2010, 49 (5), 877-
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Volatile Organic Compounds Using a Hand-Held Ion Trap Mass Spectrometer. Anal.
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organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077-
3086.
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CHAPTER 3. GAS-PHASE REACTIONS OF C3H2 WITH PROTONATED ALKYL
AMINES: FORMATION OF A C-N COVALENT BOND
“The reaction between alkyl amines and C3H2 carbenes was a surprise in the study
of bio-ion/radical reactions. However, science is sometimes not about how to follow the
design and prediction, but discovering new things others never had.”
3.1 Introduction
Vinylidene carbenes (C3H2) are unsaturated divalent carbon species displaying two
non-bonding electrons in a singlet or triplet configuration. There are three structural
isomers of C3H2: cyclopropenylidene, propadienylidene, and propynylidene. Vinylidene
carbenes overall are highly reactive and not stable in the condensed phase. However, they
have been detected in hydrocarbon flames and are considered as the key intermediates for
soot formation;1 also, cyclopropenylidene (c-C3H2), a singlet carbene, is the most
abundant cyclic hydrocarbons observed in the interstellar space.2 A significant amount of
effort has been made to synthesize relatively stable c-C3H2 derivatives.3 These derivatives
have been widely utilized as ligands of transition metal catalysts in cross-coupling
reactions.4
Given the important role of C3H2 species in the combustion chemistry, organic
synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental
understanding of their physical and chemical properties. The relative energies,
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geometries, and the isomerization among the three isomers of C3H2 have been well
mapped with the experimental gas-phase studies and theoretical calculations.5
Cyclopropenylidene is the most stable among the three isomers. Driven toward forming a
stable aromatic system (c-C3H3+) with 2 π-electrons, c-C3H2 has a gas-phase basicity (GB)
of 916 kJ/mol from experimental measurements,6 which is significantly higher than the
GB of ammonia (819 kJ/mol).7 Investigation of c-C3H2 reactivity toward organic
molecules so far has been rarely achieved by experimental methods because of its high
instability in the condense phase.3 Instead, theoretical calculations have been used to
study several types of reactions of c-C3H2 with organic molecules, including double bond
addition,8 single bond insertion,9 and skeletal rearrangement.10 In this study, we
experimentally studied gas-phase reactions of c-C3H2 with protonated alkyl amines for
the first time.
3.2 Instrumentation
Our platform (Figure 2.1a) was constructed with a DAPI-RIT-DAPI configuration
(DAPI: discontinuous atmospheric pressure interface11; RIT: rectilinear ion trap12), which
has been previously shown to provide high pressure and energetic gas flow for gas-phase
ion/molecule reactions13. The ion trap served as both reactor and mass analyzer with a
pyrolysis nozzle to generate radical species.
For general ion/radical reactions (MS scan function shown in Figure 2.1b), DAPI-1
was used to introduce alkyl ammoniums generated by nanoelectrospray (nanoESI) while
DAPI-2 connected to the pyrolysis nozzle was used to introduce vapor of carbene
precursors (placed in a glass tube with a plastic Swagelok connected to capillary 3 of 0.25
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mm i.d.). Ions of interest introduced through DAPI-1 were mass-selectively isolated and
then interacting with carbenes from the dissociated precursors by pyrolysis from DAPI-2.
A glow discharge electron impact (GDEI)14 source was installed to emit electrons into the
RIT to ionize the neutral radicals or radical precursors that are subsequently characterized
by MS analysis. This system represented a simple and intuitionistic approach to carry out
and study gas-phase reactions and show the first experimental evidence of a gas-phase
reaction between c-C3H2 and protonated ammoniums forming a C-N covalent bond.
Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical
species and (b) the general MS scan function for ion/radical reactions (y-axis: voltage).
DAPI: discontinuous atmospheric pres-sure interface; RIT: rectilinear ion trap; GDEI:
glow discharge electron impact; rf: radio frequency; ac: alternative current.
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A single-phase radio frequency (rf) was applied to trap ions at 920 kHz or 1490 kHz
for mass range of m/z 80-600 or 20-200, respectively. The control electronics was
directly adopted from a Griffin Minotaur 300 (FLIR Systems, Inc., Wilsonville, OR). The
front endcap (close to DAPI-1) was a flat electrode with a center hole of 3 mm diameter
for ion introduction, while a mesh electrode (18 x 18 mesh size, 0.015” wire diameter;
McMaster-Carr, Aurora, OH, USA) was used for the back endcap (close to DAPI-2). A
dc of 10-30 V was applied on both end electrodes to create a dc trapping potential well
along the z direction. Resonance ejection (q = 0.81), was implemented for MS scan with
a dipolar ac signal at 324 and 524 kHz for rf frequency of 920 and 1490 kHz, respectively.
The frequency of rf 1490 kHz was used for m/z 20-180 to observe low mass ions while rf
920 kHz was used for m/z 100-600. The inductance and capacitance of the rf coil were
changed to accomplish the two frequencies. The conversion dynode of the detector was
grounded. The isolation of the trapped ions was achieved by forward/backward scan.
Collision-induced dissociation (CID) was implemented by dipolar excitation with a
single-frequency ac applied between the x electrodes of the RIT. In the mechanism study,
the low mass cutoff (LMCO) of CID was set to m/z 25 for rf 1490 kHz to trap low mass
CID fragments (eg. C3H3+, m/z 39).
The pyrolysis nozzle was first developed by Peter Chen to generate intense radical
species,15 which has been an efficient means to produce and transfer intense radicals to
gas phase16. Reactive species such as ethylny, viny, allyl, phenyl radical, and carbenes
etc. were generated by resistance-heated SiC nozzle from corresponding precursors15,
whose structures were then analyzed by infrared and electronic spectra. Flowing-
afterglow selected-ion flow tube (SIFT)17 and Fourier transform ion cyclotron resonance
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(FTICR)18 mass spectrometers were the two instruments reported to study ion/radical
reactions by pyrolysis nozzle. Proton transfer, associative detachment, and radical
recombination reactions were observed when using allyl radicals with hydroniums, ortho-
benzynes with hydroxides, and allyl radicals with benzyl radical cations, respectively. In
our setup, a silicon carbide (SiC) tube of 1 cm long, 1 mm i.d., 2 mm o.d. (Hexoloy SA,
Niagara Falls, NY) was employed as the resistance heater with an alumina tube
(8746K12, McMaster-Carr, Aurora, OH, USA) for insulation. The SiC and the Al2O3
tube were nested and cemented with ceramic adhesive (903HP, Cotronics, Brooklyn, NY)
into the machinable Al2O3 flange (8484K78, McMaster-Carr, Aurora, OH, USA).
Stainless steel capillary 2 inside the pinch valve of DAPI-2 was directly inserted into the
Al2O3 tube without further sealing. Electrical contact was provided by graphite disks
(AXF-FQ Poco Graphite, Decatur, TX) and 0.127-mm tantalum-foil clips (Alfa Aesar,
Ward Hill, MA). Two light bulbs of 100 W and 200 W, parallel to each other and in
series with the nozzle, were used as the ballast. Both nanoESI and radical precursors were
protected in Helium environment ca 760 Torr to prevent the heated SiC from oxidation.
The major differences for the pyrolysis nozzle in our platform compared to those in
flowing-afterglow selected ion flow tube or Fourier transform ion cyclotron resonance is
1) the high pressure ca 10-100 mTorr in the vacuum chamber during precursor injection
and 2) injection duration of 50 ms continuously instead of 200 µs with 20-40 Hz
frequency. Therefore, the radicals produced through the pyrolysis nozzle were expected
to have 103 times more collisions in our platform than that in high vacuum (10-5 Torr) and
pyrolysis temperature was dropping during the opening. The high pressure and energetic
gas flow are the two factors contributing to the formation of nonspecific C3H2 carbene
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from various precursors. Among three structures of C3H2 carbene species, the most stable
c-C3H2 is more likely to survive under the given environment.
Multiple triggers provided by the control electronics were used to synchronize the
operations of DAPIs, GDEI and MS analysis. All the spectra presented are the average of
5-9 continuous scans.
3.3 Materials
Ionic reactants of alkyl amines and amino acids were purchased from Sigma
Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Different concentrations (5-100 µM)
of the samples were prepared with methanol for nanoESI with 0.2 % acetic acid added to
increase the intensity of protonated species if necessary.
Radical precursors including allylchloride, 1,5-hexadiene, pentane,
propargylchloride were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis,
MO, USA). Propargylbromide was purchased from TCI America (Portland, OR, USA),
and d12-pentane was purchased from Alfa Aesar (Ward Hill, MA, USA). The head-vapor
of radical precursors in Helium buffer was injected into the platform to generate radical
species. 1,3-dibromopropyne was synthesized as C3H2 carbene precursors according to
the previous papers.19 In short, 1,3-dibromopropyne was prepared in two steps: Propargyl
alcohol was converted to 1-bromopropyn-3-ol by triphenyl phosphite methiodide.20 PBr3
was used in benzene and then pyridine to replace the hydroxyl group with Br.19 The final
product was distilled under reduced pressure with Argon.
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3.4 Results and discussion
3.4.1 Carbene reaction with protonated alkylamines
We used 1,3-dibromopropyne (BrC≡CCH2Br) as a precursor for C3H2 formation by
pyrolysis.19 Figure 3.2a shows a typical reaction spectrum of protonated n-heptyl amine
(m/z 116) and the pyrolysis products of 1,3-dibromopropyne. Three new peaks, m/z 114,
154 and 192, were observed after the reaction. The ions of m/z 154 and 192 correspond to
a mass increase of 38 Da and 2 × 38 Da, respectively, from the precursor ion (m/z 116).
The peak at m/z 114 likely resulted from the dissociation of ion m/z 154 due to excitation
by hot gas flow when opening DAPI-2 for introducing pyrolysis products.13 Collisional
activation of ion m/z 154 (Figure 3.2b) yielded ion m/z 114 through a neutral loss of 40
Da (C3H4), which supported C3H2 adduct formation.
Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with
heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2 reaction of
protonated n-heptylamine with heated d12-pentane; and (d) MS2 reaction of protonated n-
heptylamine 18C6 complex with heated 1,3-dibromopropyne.
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Besides 1,3-dibromopropyne, we found that analogous reaction products, viz. the
mass increases of 38 Da, 2 × 38 Da, or even 3 × 38 Da to the protonated n-heptyl amine
ions, were obtained when using other hydrocarbons as precursors for pyrolysis, including
allylchloride, propargyl-bromide, 1,5-hexadiene, and pentane (Figure 3.3). The last
reaction allowed us to use fully deuterated pentane (d12-pentane) as a pyrolysis precursor
to confirm the chemical identity of the 38 Da adduct. The reaction spectrum is shown in
Figure 3.2c. Reaction products at m/z 156 and 196 correspond to mass increases of 40 Da
(C3D2) and 2 × 40 Da, respectively, from the amine ions (m/z 116), consistent with our
conclusion that the addition indeed results from C3H2. All the reaction and their
corresponding CID spectra of the test amines and pyrolysis precursors can be found in
Table 3.1 and Table 3.2.
Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b)
propargylbromide, (c) 1,5-hexadiene, and (d) pentane.
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We also investigated reactions of secondary, tertiary, and quaternary
alkylammonium ions with C3H2. Similar reaction phenomena were observed for all the
amine ions except for the quaternary alkylammonium ion, for which no reaction product
was observed (Figure 3.4). This series of experiments indicated that an amine nitrogen
and an ammonium proton were involved in the reaction. We further tested reactions of
C3H2 with the non-covalent complex of 18-crown-6 ether (18C6) and protonated n-heptyl
amine. In such a complex, three strong hydrogen bonds are formed between the
protonated primary amine and three oxygen atoms of 18C6, making neither the proton
nor the nitrogen available for further reaction or binding. Under the same reaction
condition previously used for the n-heptyl amine, the addition of C3H2 to the complex ion
m/z 380 ([M+18C6+H]+) was not observed (Figure 3.2d).
Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c)
cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID spectra of the
reaction products for (d) dibutylamine and (e) tributylamine.
The studies reported above suggest that protonation is an important factor for
observing the C3H2 addition reaction. In order to gain further evidence, we compared the
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C3H2 reactions with proline methyl ester ions in protonated vs. sodium adduct forms. The
most favorable protonation site of proline methyl ester is at the pyrrolidine nitrogen,
while for the sodium adduct, the sodium ion is chelated by the carbonyl oxygen and the
amine nitrogen atoms.21 The reaction course for these two ions was drastically different.
While an efficient C3H2 addition was observed for the protonated ions, the reaction yield
was extremely low for the sodium adduct ions (Figure 3.5). This observation is consistent
with the results for the alkyl amines, for which only the protonated amines undergo
efficient C3H2 additions.
Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester
with heated allylchloride, and their corresponding product MS3 CID spectra (c) and (d),
respectively.
C3H2 has three known isomeric structures, viz. cyclopropenylidene (c-C3H2),
propadienylidene, and propynylidene which is a 3A” triplet in the ground electronic
state.5d To explain the course of the ammonium ion addition, it is important to identify
which structure(s) are involved in the observed reactions. When GDEI was used to
identify the pyrolysis product(s), the different MS patterns with heating on and off (Table
3.3) prove that new species were generated through pyrolysis. The GDEI data is not
conclusive but indicative for the identity of C3H2 since extensive ion/molecule reactions
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would occur after the initial electron impact at the high pressure (10 mTorr). The fact that
a variety of hydrocarbons could all produce C3H2 suggests that the C3H2 formation might
not be closely tied to the pyrolysis chemistry; instead, it is most likely determined by the
energetics and reactions after the pyrolysis. Note that the ion trap pressure was relatively
high (10-100 mTorr) when the pyrolysis valve was open, and the free sonic expansion
was shorter than 2 cm, while the distance between the nozzle and the center of the RIT is
5 cm. The first generation pyrolysis products can undergo extensive and energetic
collisions as they are entering the ion trap,13, 22 be converted to C3H2, which subsequently
reacts with the trapped ions. Since the reaction chemistry involves the protons on the
ammonium ion, we suspect that c-C3H2, which has a relatively high GB, 916 kJ/mol at
298 K for the 1A1 state, as compared to 860 kJ/mol for (1A1) propadienylidene, and 687
kJ/mol for the 3A” state of propynylidene, might be the dominant species formed by
pyrolysis. In order to clarify the reaction mechanism, further evidence was sought from
theoretical calculations and additional experiments.
3.4.2 Theoretical calculation of the reaction mechanism
Since C3H2 has three identified isomeric structures, i.e., cyclopropenylidene (c-
C3H2), propadienylidene (l-C3H2), and propynylidene (l-C3H2), it is of importance to
know which structure(s) are involved in the observed reactions. We tested a variety of
hydrocarbons including allylchloride, 1,5-hexadiene, propargyl-bromide, and pentane as
C3H2 precursors and observed the same reaction phenomenon as shown in Figure 3.2a,
where 1,3-dibromopropyne was used as the C3H2 precursor and reacted with protonated
alkyl amine. These results suggest that the C3H2 formation is not closely tied to the
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pyrolysis chemistry or process, instead mostly determined by the energetics and reactions
after pyrolysis. Note that the ion trap pressure is relatively high (10-100 mTorr) upon the
opening of the pyrolysis valve of the current instrument design, which gives only less
than 2 cm free sonic expansion of the radical species after the initially formed from
pyrolysis in comparison with 4 cm between the nozzle and the center of the RIT. These
radical species undergo extensive and energetic collisions as they enter the trap13, 22 and
are further converted into other products. We suspect that c-C3H2 might be the dominant
species formed from such process since c-C3H2 has lower energy by about 42–59 and 59–
92 kJ/mol than that of the propadienylidene and propynylidene.1, 5a, 23 This hypothesis is
also supported by the reaction chemistry which is mainly driven by proton. Indeed, c-
C3H2 has a significant GB of 916 kJ/mol as mentioned above. A reaction mechanism of
proton-bound dimer formation between c-C3H2 and protonated alkyl amine as the first
step followed by C-N covalent bond formation was proposed to explain the observed
reaction phenomenon in Figure 3.2a. This mechanism was further investigated and
evaluated by theoretical calculations and experimental results.
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Figure 3.6 Calculated PES of (a) CH3NH3+/c-C3H2 reaction to form a C-N covalent bond
from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ, kJ/mol) and (b)
the two Brauman intermediates as a function of the distance of N-H---C, blue square:
CCSD(T)/aug-cc-pVTZ single point energies on M06-2X/6-311+G(2d,p) optimized
geometries; open circle: M06-2X/6-311+G(2d,p) optimized; filled circle: B3LYP/6-
311+G(2d,p) optimized.
The reaction between c-C3H2 and protonated methyl amine (CH3NH3+) was used as
a simplified model for theoretical calculations at the CCSD(T)/aug-cc-pVTZ//M06-2X/6-
311+G(2d,p) level of theory and including zero point vibrational energies. The key
structures involved in the reaction together with their 0 K enthalpies (relative to the
enthalpy of the reactants) are illustrated in Figure 3.6a. The first step of the reaction is the
formation of a proton-bound dimer between c-C3H2 and CH3NH3+. This step is
exothermic, producing two Brauman intermediates, Complex 1 (-127 kJ/mol) and
Complex 2 (-121 kJ/mol) that were found as the structures with the lowest energies
(Figure 3.6b). Complex 2 has a structure with a bonding distance between the proton and
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the carbon of the c-C3H2 (N-H---C = 1.16 Å), as compared to Complex 1 (N-H---C =
1.72 Å), which indicates that the proton moved from the amine nitrogen to the c-C3H2
moiety. This shift places a partial positive charge on the C1 of c-C3H2 and facilities a
subsequent nucleophilic attack by the amine nitrogen, leading to the formation of a C-N
bond in product A (-216 kJ/mol) through a low energy barrier. Product A might quickly
go through rearrangements, such as ring opening and hydrogen atom migration (TS1 in
Figure 3.6a), to form a more stable product B (-372 kJ/mol). Product B is shown with a
cis-syn geometry that can isomerize to a slightly more stable trans-isomer. In either
isomer, two of the original amine hydrogens are transferred to the C3H4 moiety. It is
worth noting that Complex 1 has the proton more tightly bound to the amine nitrogen
and, as a result, the N-C covalent bond formation has a high energy barrier (Figure 3.7).
Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A
3.4.3 Experimental evidences of the reaction mechanism
The mechanism proposed in Figure 3.6 shows that the formation of a proton-bound
dimer between c-C3H2 and protonated alkyl amine is the key step for the overall reaction.
The proton-bound dimer can stay as it is (Complex 1) or undergo further rearrangement
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to form a covalent product. Although the proton-bound dimer and the covalent product
has the same m/z (indicated as [C]), they should be distinguished via unique unimolecular
dissociation channels upon collisional activation. For instance, loss of 40 Da (m/z [C-40])
is the signature fragmentation channel from the covalent product as shown in Figure 3.2b.
CID of proton-bound dimer, however, leads to the separation of c-C3H2 and alkyl amine.
Proton transfer product, C3H3+ (m/z 39), and protonated amines (m/z [C-38]) should be
the main products, and their relative abundances are dependent on the gas-phase
basicities of the corresponding neutrals. A breakdown curve of protonated
butylamine/carbene reaction as an example is illustrated in Figure 3.8 to show the
relatively abundance of these three CID products together with the precursor complex as
a function of the resonance activation ac amplitude. The yields of three products are
relatively stable throughout the range of the activation energies, which implies that the
three products have similar energy barriers for dissociation.
Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional
activation: relative intensity of parent complex ([C], blue circle), covalent product ([C-
40], red diamond), proton-transfer product (m/z 39, green triangle), and direct
dissociation ([C-38], green square) as a function of activation energy.
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The relative intensity of [C-40] among all fragment ions can be used to indicate the
yield of the covalent product (% Covalent Complex). If the mechanism proposed in
Figure 3.6 truly reflects the nature of the reaction, % Covalent Complex could be varied
by changing the gas-phase basicity of the amines. Using the calculated GB of c-C3H2 920
kJ/mol as a bench mark (CCSD(T)/aug-cc-pVTZ single-point energies on CCSD/6-
311+G(2d,p) optimized geometries), we investigated reactions of c-C3H2 produced by
pyrolysis of allylchloride with a series of protonated alkyl amines and guanidine, with
their GBs ranging from 884 to 949 kJ/mol.7 The complexes ([C]) formed by these
reactions were subjected to CID to achieve 80-90% of dissociation.
The CID spectra of complexes formed by c-C3H2 addition to protonated n-butyl
amine, ethylmethyl amine, di-n-propylamine, and guanidine are shown in Figure 3.9a-d.
They represent cases that amines have GBs lower, comparable, higher, and significant
higher than that of c-C3H2, respectively. CID of the butylamine-C3H2 complex (m/z 112)
generates [C-40] (m/z 72) and m/z 39 at about equal relative intensity while the intensity
of [C-38] (m/z 74) is very low (Figure 3.9a). This is because the GB of n-butylamine
(887 kJ/mol) is smaller than that of c-C3H2 which is therefore more favorably protonated.
Based on the data shown in Figure 3.9a, the yield of the covalent complex is about 52%.
The CID spectrum of the ethylmethylamine-C3H2 complex (Figure 3.9b) shows the loss
of 40 Da (m/z 58) as the main fragmentation, leading to 68 % Covalent Complex. Given
the similar GBs of ethylmethylamine (909 kJ/mol) and c-C3H2, the ion intensities of
C3H3+ (m/z 39) and protonated amine (m/z 60) are comparable. When the GB is further
increased, as in the case of di-n-propylamine, (GB= 929 kJ/mol), only two fragment
channels are observed, [C-40] and [C-38], while m/z 39 intensity diminishes as a result of
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a lower GB of c-C3H2 relative to the amine. The % Covalent Complex is 49%. Guanidine
has the highest GB (949 kJ/mol)24 among all the species tested. CID of the complex
shows a loss of neutral carbene ([C-38], m/z 60) as the only fragmentation pathway.
Figure 3.9e summarizes the correlation between the % Covalent Complex and the
GB values of various alkyl amines and guanidine. The corresponding CID spectra are
shown in Table 3.2. The highest yield (82%) for the formation of a covalent product is
obtained for cyclohexylamine, which has a GB value slightly lower than c-C3H2. The
yield decreases as the GB difference becomes larger. The fact that covalent complex
formation is most favored with a minimal GB difference25 between the amines and c-
C3H2 strongly supports the formation of the proton-bound dimer is the critical step, as
pointed out by the theoretical calculations (Figure 3). Furthermore, it also supports that c-
C3H2 instead of other two isomers is mainly responsible for the reaction. The 1A1 ground
state of propadienylidene is both substantially less stable (+53 kJ/mol) and less basic (GB
= 860 kJ/mol) than c-C3H2. Propynylidene exists as a high-energy triplet state (3A”, +47
kJ/mol) of low basicity (GB = 687 kJ/mol) when forming a linear triplet C3H3+ cation.
90
90
Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b)
ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and (e) %Covalent
of protonated alkyl amines/c-C3H2 reactions as a function of GBs of the corresponding
alkyl amines. The %Covalent is calculated as I[c-40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40],
and I[c-38] correspond to the intensity of m/z 39 as the proton-transfer product, neutral loss
of 40 Da as the covalent product, and neutral loss of 38 Da as the direct-dissociation
product, respectively, in the MS3 CID spectra based on 80-90% parent ions fragmented.
Error bars were based on 5 duplicates of an average of 3 spectra.
We also formed amine-C3H2 proton-bound dimer from reactions of neutral amines
(i.e. heptylamine) and C3H3+ ions. Ethyl, propyl, butyl, hexyl and heptyl amines were
investigated for ion/molecule reaction with c-C3H3+ ions. Table 1.1 summarized the
ion/molecule reaction spectra and their corresponding MS3 CID spectra. In comparison
with corresponding ion/carbene reactions, complexes of exactly the same m/z values were
observed for the reactions with neutral amines. Collisional activation of the heptylamine-
91
91
C3H3+ complex (Figure 3.10) produced a very similar fragmentation pattern to that of the
C3H2-protonted heptyl amine product shown in Figure 3.2b. The above result also
supports the reaction mechanism with the proton-bound dimer intermediate suggested for
the ion-carbene reactions.
Figure 3.10 MS3 reaction product CID spectrum of C3H3+and neutral n-heptyl amine
3.5 Conclusion
In summary, the home-developed DAPI-ion trap-pyrolysis MS platform facilitated
studies of reactions of c-C3H2 with protonated alkyl amines in the gas phase. Addition of
C3H2 to the protonated amine was observed as the only reaction channel. Mechanistic
studies demonstrate that non-covalent and covalent (via C-N formation) complexes can
be produced and they both stem from the formation of proton-bound dimers as the key
step. The covalent product formation is in favor of alkyl amines with similar GB values
as that of the c-C3H2. In the interstellar carbon chemistry, it has been long believed that
the major pathway for the formation of c-C3H2 comes from electron attachment to c-
C3H3+,26 while the major pathway for the destruction of c-C3H2 goes through the
protonation of c-C3H2 to form c-C3H3+, which makes c-C3H2 a “dead end” in this carbon
cycle.26-27 This study therefore provides a new perspective to other possible reaction
92
92
pathways of c-C3H2 in the interstellar medium. In the presence of protonated amines or
even ammonium, c-C3H2 can form covalent adducts to seed the formation of larger
organic molecules/ions consisting of C, N, and H atoms.
3.6 Appendix
Table 3.1 summarizes MS2 reaction spectra of all alkyl ammoniums investigated
with C3H2 generated by the pyrolysis of various organic precursors. The number of +38
Da adducts was corresponding to the number of protons on the charged amine site. The
nonspecific generation of C3H2 carbenes was observed from precursors with three or
more hydrocarbons. Therefore, cyclopropenylidene was most-likely the carbene species
produced during pyrolysis because it is more stable compared to the other two C3H2
carbenes. The series of adducts could be both covalent products and simple complexes,
thus CIDs were then used for structure elucidation on the selected carbene reaction
products.
Table 3.1 Ammonium reactions with various radical precursors
Reactant Radical
Precursor MS Spectrum No.
Heptylamine
[M+H]+
m/z 116
BrC≡CCH2Br
1,3-
Dibromopropyne
1
Allylchloride
2
NH2
Cl
93
93
Allylbromide
3
Allyliodide
4
Propargylbromide
5
1, 5-Hexadiene
6
Pentane
7
Pentane-D12
8
Heptylamine
18-Crown-6
[M+18C6+H]+
m/z 380
Propargylbromide
9
Heptylamine
12-Crown-4
[M+18C6+H]+
m/z 292
Propargylbromide
10
Br
I
Br
D3C
D2C
CD2
D2C
CD3
NH2
Br
NH2
Br
94
94
Heptylamine H/D
[M-2H+3D]+
m/z 119
1, 5-Hexadiene
11
Methylamine
[M+H]+
m/z 32
Propargylbromide
12
Ethylamine
[M+H]+
m/z 46
1, 5-Hexadiene
13
Propylamine
[M+H]+
m/z 60
Allylchloride
14
Butylamine
[M+H]+
m/z 74
Allylchloride
15
Isobutylamine
[M+H]+
m/z 74
Allylchloride
16
Hexylamine
[M+H]+
m/z 102
Allylchloride
17
Cyclohexylamine
[M+H]+
m/z 100
Allylchloride
18
ND
DD
Br
Cl
Cl
Cl
Cl
Cl
95
95
Ethylmethylamine
[M+H]+
m/z 60
Allylchloride
19
Diethylamine
[M+H]+
m/z 74
Allylchloride
20
Dipropylamine
[M+H]+
m/z 102
Allylchloride
21
Dibutylamine
[M+H]+
m/z 130
Allylchloride
22
Propargylbromide
23
1, 5-Hexadiene
24
Dibutylamine H/D
[M-H+2D]+
m/z 132
Propargylbromide
25
Diisopropylamine
[M+H]+
m/z 102
Allylchloride
26
Cl
Cl
Cl
HN
Cl
Br
NDD
Br
Cl
96
96
Guanidine
[M+H]+
m/z 60
Allylchloride
27
Tributylamine
[M+H]+
m/z 186
Allylchloride
28
Propargylbromide
29
1, 5-Hexadiene
30
Tributylamine H/D
[M+D]+
m/z 187
Propargylbromide
31
Cetyltrimethyl
ammonium
M+
m/z 284
Propargylbromide
32
1, 5-Hexadiene
33
Ion-trap CID for the carbene adducts was performed (shown in Table 3.2) to
illustrate three reaction channels discussed in 3.4.3. For the data points in Figure 3.9e of
%Covalent complex vs. GB curve, a LMCO of m/z 25 was used for all MS2 reactions and
Cl
N
Cl
Br
N
D
Br
N
CH3
CH3
CH3
H3C(H2C)15
Br
97
97
MS3 CIDs with the rf frequency of 1490 kHz. The rf frequency of 920 kHz was used to
trap ions larger than m/z 100 and to record the rest reaction and CID spectra. The final
stable product shown in Figure 3.6 had two of the original hydrogens of the carbene side,
so that deuterium scrambling was expected in MS3 CID spectra when using d12-pentane
as the carbene precursor (CID No.44) and H/D exchanged heptyl ammonium (CID
No.45).
Table 3.2 MS3-MS5 CID spectra of selected reaction products
Reaction No. Selected CID
precursor m/z MS Spectrum CID No.
1 MS3
116-154
34
2 MS3
116-154
35
2 MS3
116-192
36
2-36 MS4
116-192-154
37
2 MS3
116-230
38
98
98
2-38 MS4
116-230-192
39
2-38-39
MS5
116-230-192-
154
40
5 MS3
116-154
41
6 MS3
116-154
42
7 MS3
116-154
43
8 MS3
116-156
44
11 MS3
116-157
45
13 MS3
46-84
46
99
99
14 MS3
60-98
47
15 MS3
74-112
48
16 MS3
74-112
49
17 MS3
102-140
50
18 MS3
100-138
51
19 MS3
60-98
52
20 MS3
74-112
53
21 MS3
102-140
54
100
100
22 MS3
130-168
55
22 MS3
130-206
56
22-56 MS4
130-206-168
57
26 MS3
102-140
58
27 MS3
60-98
59
28 MS3
186-224
60
The internal ion source GDEI was designed to show the MS characterization with
pyrolysis on or off in Table 3.3. However, the radical precursors are usually easy to
generate reactive ion species (eg. C3H3+)28 by electron impact ionization. Based on the
pressure of 10-100 mTorr after DAPI opening, further reactions would occur between
ions and neutral precursors (also the impurity in the precursor) at the number density of
101
101
3.3-33x1014 cm-3. Therefore, the GDEI spectra could not directly reflect the radical
species generated by pyrolysis. Since the reactions between alkyl ammoniums and
radicals from various precursors to form +38 covalent adducts have strongly supported
the generation of C3H2 carbene species by the pyrolysis nozzle, the purpose of GDEI
characterization was just to observe the pattern changing when the pyrolysis nozzle was
turned on or off. The changing of GDEI pattern between room temperature and heated
precursors indicated the formation of new species by pyrolysis for all the precursors,
which was considered an indirectly evidence of C3H2 carbene reaction.
Table 3.3 GDEI spectra of radical precursors Precursor Pyrolysis MS Spectrum
BrC≡CCH2Br
1,3-Dibromopropyne
Off
On
Allylchloride
Off
On
Cl
102
102
Allylbromide
Off
On
Allyliodide
Off
On
Propargylbromide
Off
On
1, 5-Hexadiene
Off
On
Br
I
Br
103
103
Pentane
Off
On
C3H3+ ion/molecule reactions have been previously studied with multiple reaction
channels observed such as interactions with unsaturated hydrocarbons,28-29 proton transfer
and complexes.30 However, no CID results were reported to study the final structure of
the products, probably due to the instrumentational setup. In this section, we used
propargylchloride to generate a majority of c-C3H3+ ions by GDEI as comparison to the
proposed c-C3H2 carbene reactions. It was previously shown that propargylchloride
produced 85-90% of non-reactive cyclic C3H3+ ions by electron impact or chemical
ionization.28 No effort was made to further separate linear (reactive) and cyclic (non-
reactive) C3H3+ species. After isolating C3H3
+ m/z 39 ions with rf 1490 kHz at a LMCO of
m/z 10, neutral amines were injected through DAPI-1 with an additional capillary. The
reaction took place at LMCO of m/z 25, which allowed the trapping of m/z 39-180. CIDs
were performed at LMCO of m/z 25 as well. The MS3 CID spectra of ion/molecule
reactions in Table 1.1 were very similar to those of ion/carbene reactions in Table 3.2,
which proved they shared common reaction mechanism by forming a proton-bound
dimer.
104
104
Table 3.4 Ion/molecule reactions of c-C3H3+ and neutral amines
Reactants MS type MS Spectrum
c-C3H3+
+
Ethylamine
MS2
39-
MS3 CID
39-84-
c-C3H3+
+
Prpylamine
MS2
39-
MS3 CID
39-98-
c-C3H3+
+
Butylamine
MS2
39-
MS3 CID
39-112-
c-C3H3+
+
Hexylamine
MS2
39-
MS3 CID
39-140-
105
105
c-C3H3+
+
Heptylamine
MS2
39-
MS3 CID
39-154-
106
106
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reaction mechanism of cyclopropenylidene with azacyclopropane: ring expansion process.
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109
109
CHAPTER 4. GAS-PHASE C-C3H2 CARBENE REACTIONS WITH
BIOMOLECULAR IONS
4.1 Introduction
It has been discussed in the previous chapter that c-C3H2 can be generated by non-
specific pyrolysis under the conditions in DAPI-RIT-DAPI mass spectrometer with 10-2
Torr pyrolysis pressure and energetics gas flows. The reactions between protonated alkyl
amines and c-C3H2 have been studied and covalent products have been observed to form
through a proton-bound dimer mechanism with further rearrangements.
Biomolecules such as nucleobases, nucleosides, amino acids, peptides, and proteins
are more complicated than alkyl amines, which also contain amine functional groups. The
protonated biomolecules are expected to be modified by c-C3H2 carbene through proton-
bound dimer, possibly to form covalent reaction products. Besides, c-C3H2 carbene have
been theoretically calculated to react with a single R-H (R=F, O, N) bond for insertion
and also with C=O and C=C bonds for addition.1 Furthermore, reactive functional groups
including thiyl group, disulfide linkages are present in amino acids and peptides. These
functional groups were reported to be reactive with free radicals like hydroxyl radicals
and sulfinyl radicals.2 It is still unknown if the bi-radical species c-C3H2 could modify
these functional groups in their gas-phase ionic forms. In this chapter, the reactivity of c-
C3H2 carbene was studied by the interaction with protonated biomolecules mentioned
above for proton-bound reactions as well as other possible gas-phase reactions.
110
110
4.2 Experimental section
Materials
All nucleobases, nucleosides, amino acids, peptides, proteins and lipids were
purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different
concentrations (5-100 μM) of the samples were prepared with methanol/water (9:1, v/v)
with 1 % acetic acid added to increase the intensity of protonated species if necessary.
Radical precursors including allylchloride and 1,5-hexadiene were also purchased from
Sigma. Allylchloride was chosen as the major C3H2 carbene precursor unless otherwise
specified.
MS System
All conditions were directly adapted from Chapter 3 except for the rf frequency. Two
rf frequencies were utilized: the frequency of 950 kHz was employed for m/z 100-500 to
observe reactions of amino acids, small peptides, nucleobases and nucleosides, whereas
the frequency of 650 kHz allowed high mass range to m/z 1500 to analyze ions of large
peptides and proteins. Resonance ejection (q = 0.81) was implemented for MS scan with
a dipolar ac signal at 334 and 229 kHz for rf frequency of 950 and 650 kHz, respectively.
4.3 Results and discussion
4.3.1 Nucleobases and nucleosides
The interactions of protonated nucleobases and nucleosides (structures and gas-phase
basicities shown in Table 4.1) with c-C3H2 carbene generated by pyrolysis of
allylchloride have been investigated to study the carbene modification of DNA/RNA
related biomolecules in gas phase.
111
111
Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4
Nucleobase Structure
Gas-
phase
basicity
(kJ/mol)
Nucleoside Structure
Gas-
phase
basicity
(kJ/mol)
Adenine
912 Adenosine
957
Guanine
928 Guanosine
961
Cytosine
918 Cytidine
950
Uracil
842 Uridine
917
Thymine
850 Thymidine
916
For nucleobases of adenine, guanine, and cytosine, their GBs are comparable to that
of c-C3H2 carbene.5 Thus, the MS2 carbene reaction spectra were very similar for these
three nucleobases and a significant amount of +38 Da adducts were observed. Taking
adenine reaction as an example shown in Figure 4.1a, mass increases from 1 × 38 Da to 3
× 38 Da; which corresponded to mono-, di-, and tri- carbene adducts were observed.
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112
Despite adenine having multiple protonation sites,6 the 3 × 38 Da mass increase of m/z
250 implied a primary amine was responsible for protonation in this specific reaction. In
the MS3 CID spectrum of Figure 4.1b, a direct loss of 38 Da was obtained when
activating the mono-adduct of m/z 174, but the intensity was very low. According to the
close GBs of adenine and c-C3H2 carbene,7 a covalent bond formation should be favored
as experimentally demonstrated in Chapter 3. However, the adjacent carbon of the
primary amine on adenine is not attached to a hydrogen. The covalent product therefore,
would not be able to undergo a loss of 40 Da in the fragmentation pathway as in the cases
of amines. The low yield of CID might serve as a piece of indirect evidence of the
covalent product formation. As for its corresponding nucleoside adenosine, multiple +38
Da adducts as well as the fragments from the carbene products were obtained in Figure
4.1c. Nucleosides have higher GBs than their corresponding nucleobases.8 Simple
complexes should be preferred for adenosine with GB of 957 kJ/mol. In the MS3 CID
spectrum of the reaction product at m/z 306, only a small portion of 38 Da loss (m/z 268)
appeared as the direct loss of C3H2 in Figure 4.1d, while the principle fragment was the
loss of ribose ring (loss of 132 Da). This result indicated that the hydrogen bond in the
proton-bound dimer is stronger than the covalent N-glycosidic bond. The input energy
might be able to trigger further rearrangement of the complex to form covalent product.
A very low amount of m/z 136 also appeared and this was probably due to the further
activation of m/z 174. Similar reaction phenomena were observed for nucleobases of
guanine, cytosine and nucleosides of guanosine, cytidine. All MS2 reaction and MS3 CID
spectra are included in Table 4.2.
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Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated
adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d) adenosine
carbene monoadduct
Uracil and thymine have significantly lower GBs compared to c-C3H2,9 which makes
them harder for the formation of proton-bound dimers. The major product of the
protonated molecules for these two species was expected to be a proton-transfer product
c-C3H3+, when encountering gas-phase c-C3H2 carbene. Unfortunately, ions of m/z 39
could not be trapped under rf frequency of 950 kHz. Figure 4.2a shows the MS2 reaction
spectrum of protonated uracil with c-C3H2 carbene. No adduct was formed with 38 Da
mass increase, instead only an intensity decrease of the molecular ions was recorded,
probably because of the proton-transfer reaction mentioned above. Although protonated
uracil did not form proton-bound dimer with c-C3H2 due to the low GB, its corresponding
nucleoside uridine showed such a reaction channel in Figure 4.2b since the ribose ring
pushed the electron cloud and thus increased the GB to the same level of c-C3H2.10 The
protonation might locate on the amide amine with two exchangeable hydrogens to induce
a di- carbene adduct. The MS3 CID (Figure 4.2c) of the mono-adduct in uridine reaction
yielded a similar fragmentation pathway as those of adenosine, guanine, and cytidine.
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114
The fact that uracil did not form proton-bound dimer at m/z 151 while uridine-carbene
proton-bound dimer yielded m/z 151 during CID, indicates that the formation of proton-
bound dimer could be manipulated for low GB compounds with the modification of an
easy-to-cleave side chain. For thymine and thymidine, almost identical reactions were
recorded in Table 4.2 as uracil and uridine, except that thymidine has a 2’-deoxyribose
ring instead of a ribose ring. Thus, the neutral loss observed is 116 Da instead of 132 Da
upon activation.
Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated
uridine; and MS3 CID spectrum (c) uridine carbene monoadduct
The reactions between nucleobases, nucleosides and pyrolysis-produced carbene
showed that c-C3H2 was able to modify DNA/RNA related gas-phase biomolecular ions.
The reactions of adenine, guanine and cytosine with c-C3H2 tend to form covalent
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115
products, yet the products lack dissociation pathway to form fragments during MS3 CID.
Uracil and thymine have GBs that are too low to form proton-bound dimers, however
uridine and thymidine are able to form proton-bound dimers and ribose or deoxyribose
ring loss upon CID was observed for the product ions. Adenosine, guanosine, and
cytidine can form proton-bound dimer with c-C3H2. The proton bound energy is higher
than the N-glycosidic bonds and further rearrangements might happen to covert those
dimers into covalent products.
Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 Reactants MS type MS Spectrum
Protonated
adenine
+
c-C3H2
MS2 Reaction
MS3 CID
Protonated
guanine
+
c-C3H2
MS2 Reaction
MS3 CID
Protonated
cytosine
+
c-C3H2
MS2 Reaction
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116
MS3 CID
Protonated uracil
+
c-C3H2
MS2 Reaction
Protonated
thymine
+
c-C3H2
MS2 Reaction
Protonated
adenosine
+
c-C3H2
MS2 Reaction
MS3 CID
Protonated
guanosine
+
c-C3H2
MS2 Reaction
MS3 CID
Protonated
cytidine
+
c-C3H2
MS2 Reaction
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117
MS3 CID
Protonated uridine
+
c-C3H2
MS2 Reaction
MS3 CID
Protonated
thymidine
+
c-C3H2
MS2 Reaction
4.3.2 Amino acids, peptides, proteins, and lipids
4.3.2.1 Protonated amino acids and singly charged peptides
Protonated amino acids containing both amine and carboxylic acid functional groups
are expected to react with C3H2 via the proton-bound dimer mechanism proposed in the
previous chapter. Compared to the simple amine system studied previously, gas-phase
chemistry should be more complicated with these basic building blocks of macro
biomolecules in terms of fragmentation pathways during CID.
For basic amino acids of Lys, Arg, and His, their GB values are all higher than c-
C3H2, however the MS2 reaction spectra of those three amino acids all showed abundance
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38 Da mass increases and new fragmentation pathway in MS3 CID. This was probably
due to proton relocation in the basic amino acid ions. The cyclopropenylidene would
interact at the proton site which has a similar GB as itself and form a proton-bound dimer.
For instance, the reaction in Figure 2.1a between protonated Lys and c-C3H2 produced
mono- and di- adduct of m/z 185 and 223 as well as some other fragment peaks due to the
excessive internal energy generated by this reaction. MS3 CID of the reaction product of
m/z 185 (Figure 2.1b) yielded all the fragments appeared in the reaction spectrum. A
reaction scheme is presented in Figure 2.1c to illustrate a possible fragmentation pathway
of the proton-bound dimer. The reaction product of m/z 185 could dissociate into the
original reactant protonated Lys of m/z 147 by directly losing c-C3H2 of 38 Da. Thus,
protonated Lys further underwent a NH3 loss, generating m/z 130 cyclic ions upon
activation, which was a common neutral loss for Lys and other amino acids.11 Another
fragmentation pathway was through a covalent product after the carbene reaction. Gas-
phase c-C3H2 could be covalently attached to a nitrogen atom and form a new species.
Although the location of the attachment (side chain or N-terminal) is still unknown, the
covalent product would undergo a NH3 loss similar to the protonated Lys and yield a
cyclic CID product of m/z 168 with an alkyl chain covalently bonded to the amine. This
structure could lose 40 Da C3H4 by the attack of the hydrogen on the adjacent carbon as
the fragmentation pattern of simple amines in the previous chapter, generating a cyclic
iminium of m/z 128. An alternative pathway was for the alkyl chain to grab the hydrogen
on the carboxyl acid to further induce a CO2 (44 Da) loss to form a fragment of m/z 124.
Both (1) and (2) pathways were likely to be electrophilic attacks. The loss of 44 Da did
not appear in low energy fragmentation pathways12 of amino acids and was highly
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possible to be related to the carbene chemistry. Indeed, similar loss was also observed in
His/carbene spectrum (Table 1.1).
Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum
of Lys/carbene reaction product, and (c) proposed fragmentation pathway of the reaction
product.
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In the case of Arg, CID of protonated Arg (m/z 175) yielded m/z 158 (-H2O), 157 (-
NH3), 140 (-H2O-NH3), 130 (-NH3-CO or -COOH), 116 (-(NH2)2C=NH), and 112 (-H2O-
NH3-CO) (Table 1.1). Therefore, the mono-adduct of m/z 213 in Arg/carbene reaction in
Figure 4.4b yielded: m/z 175 (-C3H2), 169 (-CO2), 152 (-CO2-NH3), 114 (-(NH2)2C=NH -
C3H4), 112 (-C3H2 - H2O-NH3-CO), and 110 (-C3H4 - H2O-NH3-CO) during CID. The
fragments of m/z 175 were due to the direct loss of the carbene from the reaction
monoadduct; m/z 169 and 152 confirmed the CO2 loss pattern by the carbene similar to
Lys and His; and m/z 114 and 110 were possibly the oxidized products of m/z 116 and
112 (two hydrogen removed) in the Arg CID spectrum respectively. The covalent
addition of Arg/carbene reaction might be located at the N-terminal instead of the side
guanidine chain according to the CID fragmentation pathways. However, the most
favorable protonation site was at the Arg residue in singly charged peptide Met-Arg-Phe-
Ala (MRFA) so that the MS3 CID of the complex of singly charged MRFA showed only
dominant fragment at m/z 524 by directly losing C3H2. The alternative N-terminal of Met
residue was unlikely to be protonated.
Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged
MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d) MRFA/carbene
reaction product.
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121
The results of protonated basic amino acids and MRFA interacting with
cyclopropenylidene show that although the GBs of the ionic reactants were much higher
than C3H2, covalent products could still be formed based on the MS3 CID fragments, as
long as an alternative protonation site existed close enough to the favored charged
position in the ionic species which had a suitable GB to form a proton-bound dimer for
further rearrangements. Other amino acids were also tested for their modifications with
C3H2 carbene and shown in Table 1.1. A neutral loss of 46 Da (-H2O-CO) was observed
for Phe/C3H2 reaction products;13 a loss of 40 Da (-C3H4) was found for Tyr/C3H2
reaction products; a loss of 17 Da (-NH3) was obtained for Met/C3H2 and Gln/C3H2
reaction products. These results support the formation of a covalent bond.
4.3.2.2 Multiply charged peptides and proteins
Multiply charged peptides could facilitate the C3H2 complex formation through the
proton-bound dimer mechanism due to more charges buried inside one ionic species.
Indeed, triply charged Angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,
DRVYIHPFHL) of m/z 433 had up to ten adducts when encountering gas-phase C3H2 by
the pyrolysis of allylchloride in Figure 4.5. The number of adducts was related to the
number density of C3H2 carbene generated by pyrolysis, which is as a function of the
pyrolysis temperature. For 750 degree Celsius, the precursor triply charged ions together
with up to four carbene adducts appeared in carbene reaction spectrum (Figure 4.5a),
whereas no precursor ions were present anymore for the pyrolysis temperature of 800 and
900 degree Celsius in Figure 4.5b and c. With the higher temperature for pyrolysis, up to
ten carbene adducts were observed in Figure 4.5c.
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Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride
at (a) 750, (b) 800, and (c) 900 degree Celsius.
Multiply charged protein can also react with the gas-phase carbene species. No mass
selection was made to isolate a single ionic population. The purpose of this set of
experiment was to evaluate the carbene reactivity towards different charge states. For
Ubiquitin, seven to twelve multiply charged proteins were observed in MS1 full scan in
Figure 4.6a. The protein-carbene complexes corresponding to the eight to eleven charge
states were formed in Figure 4.6b. The number of carbene adducts has an increasing trend
with the increase of charge states as predicted. The 11+ charge state species yielded
approximately 35 carbene adducts based on the difference of m/z values. The lowest
addition number observed in this experiment was ca. 23 for the 8+ charge state.
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123
Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its
reaction with heated allylchoride.
4.3.2.3 Reactive functional groups
Since carbene is a highly reactive species, it would be interesting to investigate if this
bi-radical species would interact with relatively reactive functional group such as thiyl
group in Cys and disulfide linkages in peptides. Acetyl cysteine with the N-terminal
blocked was selected to test the reactivity of thiyl group and c-C3H2 carbene, which was
designed to minimize the reaction of the protonated amine site. The very low yield of 38
Da mass increase from the protonated species in Figure 4.7a, proved that thiyl group had
limited reactivity when encountering gas-phase c-C3H2 carbene. The mass increase was
most likely due to the complex formation with the protonated amide, similar to the cases
in thymine and uracil.
Disulfide linkage is an important posttranslational modification to construct three
dimensional structure in proteins. A simple disulfide linked di- amino acid cystine was
used in its protonated form to interact with c-C3H2 carbene. Despite the abundant 38 Da
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mass increases in the reaction spectrum (Figure 4.7b), there was no ion observed from
m/z 80-160 (cysteine thiyl radical ion: m/z 121, perthiyl radical ion: m/z 153), which
indicated no disulfide cleavage occurred during the gas-phase carbene reaction. Moreover,
using singly charged oxidized glutathione (single chain sequence: γ-Glu-Cys-Gly, γECG,
disulfide linked), the m/z shift still appeared to be 38 Da, however, no sign of disulfide
cleavage was observed in Figure 4.7c.
Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated
cystine, and (c) singly charged oxidized glutathione.
Unsaturated phospholipids play an important role in biological functions.14 It was
theoretically calculated and reported that c-C3H2 can react with hydroxyl group for single
bond insertion,1a and neutral unsaturated compounds for double bond addition.1c, 1d
Nevertheless, there has yet to be any experimental evidence for the mentioned
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125
neutral/carbene or ion/carbene reactions. Choline was then used to test the reactivity of c-
C3H2 with hydroxyl group. The advantage of gas-phase choline ion is that it has a fixed
charge to eliminate the proton-bound dimer reaction. In the reaction spectrum of Figure
4.8a, no mass shift was observed for choline ions, inferring insertion reactions did not
occur under the current experimental conditions between choline and c-C3H2.
Furthermore, the sodiated lysophosphatidylcholine (LPC) with an eighteen-carbon acetyl
chain and two double bonds in the 9th and 12th positions (18:2∆9, 12) was generated and
isolated to interact c-C3H2 carbene in Figure 4.8b. The lack of ions at m/z 590 indicated
the gas-phase cyclopropenylidene did not undergo double bond insertion in the LPC
studied.
Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double
bond containing LPC (18:2) sodium adduct. No reaction product was observed.
The absence of reactivity of gas-phase c-C3H2 towards neutral thiyl group, disulfide
linkages, hydroxyl group, and double bonds based on the results of Figure 4.7 and Figure
4.8, suggested that the gas-phase chemistry of ion/c-C3H2 was mainly driven by the
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126
proton and the mechanism proposed in the previous chapter. The theoretically calculated
reaction pathway might not occur or be totally shadowed because of the high GB of the
cyclic carbene. All MS2 reaction and MS3 CID spectra are attached in Table 1.1 for all
the biomolecular ions studied in this section.
Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 Reactant and GB MS type MS Spectrum
Protonated Lysine
951 kJ/mol
MS2 Reaction
MS3 CID
Protonated Histidine
950 kJ/mol
MS2 Reaction
Protonated Arginine
1007 kJ/mol
MS2 Reaction
MS3 CID
MS2 CID
127
127
Protonated Leucine
881 kJ/mol
MS2 Reaction
Protonated Methionine
902 kJ/mol
MS2 Reaction
Protonated
Phenylalanine
889 kJ/mol
MS2 Reaction
MS3 CID
Protonated Tyrosine
892 kJ/mol
MS2 Reaction
MS3 CID
Protonated Glutamine
900 kJ/mol
MS2 Reaction
MS3 CID
128
128
Protonated Proline
methyl ester
MS2 Reaction
MS3 CID
Proline methyl ester
sodium adduct
MS2 Reaction
MS3 CID
Protonated Cysteine
methyl ester
MS2 Reaction
Protonated Acetyl
Cysteine
MS2 Reaction
Protonated Acetyl
Cysteine methyl ester
MS2 Reaction
Protonated Cystine
MS2 Reaction
with heated
allylchloride
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129
MS3 CID
MS2 Reaction
with heated
propargylbromide
MS2 Reaction
with heated
pentane
MS2 Reaction
with heated d12-
pentane
MS2 Reaction
with heated
dichloromethane
Oxidized glutathione
γ-ECG
|
γ-ECG
Singly charged
MS2 Reaction
Doubly charged
MS2 Reaction
Singly charged
Met-Arg-Phe-Ala
MRFA
MS2 Reaction
130
130
MS3 CID
Triply charged
Angiotensin I
Asp-Arg-Val-Tyr-Ile-
His-Pro-Phe-His-Leu
DRVYIHPFHL
MS2 Reaction
Low amount of
C3H2
MS2 Reaction
Medium amount
of C3H2
MS2 Reaction
High amount of
C3H2
Ubiquitin
MS1
MS2 Reaction
Choline
MS2 Reaction
Lysophosphatidylcholine
LPC(18:2) sodium
adduct
MS2 Reaction
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131
4.4 Conclusion
The interaction between biomolecular ions and c-C3H2 have been investigated.
Proton-bound dimers were formed for protonated nucleobases and nucleosides including
adenine, guanine, cytosine, adenosine, guanosine, cytidine, thymidine, and uridine. The
hydrogen bond energy in the proton-bound dimer was determined to be higher than the
N-glycosidic bonds in all nucleosides. The GB values for uracil and thymine are too low
for the complex to form. Protonated basic amino acids were shown to generate covalent
reaction product with c-C3H2 carbene by relocating the proton to a charged site of a
similar GB. Multiply charged peptides and proteins have a wide range of adduct
distribution with different amounts of c-C3H2 generated by various pyrolysis
temperatures. However, the gas-phase reactivity of c-C3H2 carbene was mainly driven by
the charge and high GB of this species. The bi-radical carbene species had limited or no
modification for free thiyl group and disulfide linkages. Furthermore, the calculated
reactivity of double bond addition and single bond insertion did not occur with
biomolecular ions such as choline and LPC. The proton-bound dimer reaction remained
the dominant channel for all the biomolecular ion/c-C3H2 reactions.
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4.5 References
1. (a) Jing, Y.; Liu, H.; Wang, H. L.; Yu, Y.; Tan, X. J.; Chen, Y. G.; Zhang, Y. L.;
Gu, J. S., Quantum Mechanical Study of the Insertion Reaction between
Cyclopropenylidene with R-H (R=F, Oh, Nh2, Ch3). J. Chil. Chem. Soc. 2013, 58 (4),
2218-2221; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, M. Y.; Chen, Y.
G., Theoretical study on the reaction mechanisms between propadienylidene and R-H
(R=F, OH, NH2, CH3): an alternative approach to the formation of alkyne. Struct. Chem.
2013, 24 (1), 33-38; (c) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, G. R., A
Theoretical Study on the Mechanism of the Addition Reaction between
Cyclopropenylidene and Ethylene. J. Chil. Chem. Soc. 2012, 57 (2), 1174-1177; (d) Wass,
D. F.; Hey, T. W.; Rodriguez-Castro, J.; Russell, C. A.; Shishkov, I. V.; Wingad, R. L.;
Green, M., Cyclopropenylidene carbene Ligands in palladium c-n coupling catalysis.
Organometallics 2007, 26 (19), 4702-4703.
2. (a) Durand, K. L.; Ma, X.; Xia, Y., Intra-molecular reactions between cysteine
sulfinyl radical and a disulfide bond within peptide ions. Int. J. Mass spectrom. (In press);
(b) Ma, X. X.; Love, C. B.; Zhang, X. R.; Xia, Y., Gas-Phase Fragmentation of [M + nH
+ OH](n center dot+) Ions Formed from Peptides Containing Intra-Molecular Disulfide
Bonds. J. Am. Soc. Mass Spectrom. 2011, 22 (5), 922-930; (c) Stinson, C. A.; Xia, Y.,
Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an
electrospray plume. Analyst 2013, 138 (10), 2840-2846; (d) Durand, K. L.; Ma, X. X.;
Xia, Y., Intra-molecular reactions as a new approach to investigate bio-radical reactivity:
a case study of cysteine sulfinyl radicals. Analyst 2014, 139 (6), 1327-1330.
3. Haynes, W. M., CRC Handbook of Chemistry and Physics 94th Edition Internet
Version. http://www.hbcpnetbase.com/: 2014.
4. Hunter, E. P. L.; Lias, S. G., Evaluated gas phase basicities and proton affinities
of molecules: An update. J. Phys. Chem. Ref. Data 1998, 27 (3), 413-656.
5. Wu, Q.; Cheng, Q.; Yamaguchi, Y.; Li, Q.; Schaefer, H. F., III, Triplet states of
cyclopropenylidene and its isomers. J. Chem. Phys. 2010, 132 (4).
6. de Meijere, A.; Kozhushkov, S. I., An evolving multifunctional molecular
building block: Bicyclopropylidene. Eur. J. Org. Chem. 2000, (23), 3809-3822.
7. Marek, I.; Simaan, S.; Masarwa, A., Enantiomerically enriched cyclopropene
derivatives: Versatile building blocks in asymmetric synthesis. Angew. Chem., Int. Ed.
2007, 46 (39), 7364-7376.
8. Dookeran, N. N.; Yalcin, T.; Harrison, A. G., Fragmentation reactions of
protonated alpha-amino acids. J. Mass Spectrom. 1996, 31 (5), 500-508.
9. Yao, C.; Syrstad, E. A.; Tureček, F., Electron transfer to protonated beta-alanine
N-methylamide in the gas phase: An experimental and computational study of
dissociation energetics and mechanisms. J. Phys. Chem. A 2007, 111 (20), 4167-4180.
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10. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji,
H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.;
Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; ;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.;
Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J.
C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.;
Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox,
D. J., Gaussian 09, Revision A. 02; Gaussian, Inc., Wallingford, CT 2009.
11. Marion, N.; Nolan, S. P., Well-Defined N-Heterocyclic Carbenes-Palladium(II)
Precatalysts for Cross-Coupling Reactions. Acc. Chem. Res. 2008, 41 (11), 1440-1449.
12. Mebel, A. M.; Jackson, W. M.; Chang, A. H. H.; Lin, S. H., Photodissociation
dynamics of propyne and allene: A view from ab initio calculations of the C3Hn (n=1-4)
species and the isomerization mechanism for C3H2. J. Am. Chem. Soc. 1998, 120 (23),
5751-5763.
13. Vazquez, J.; Harding, M. E.; Gauss, J.; Stanton, J. F., High-Accuracy
Extrapolated ab Initio Thermochemistry of the Propargyl Radical and the Singlet C3H2
Carbenes. J. Phys. Chem. A 2009, 113 (45), 12447-12453.
14. Wass, D. F.; Haddow, M. F.; Hey, T. W.; Orpen, A. G.; Russell, C. A.; Wingad, R.
L.; Green, M., Cyclopropenylidene carbene ligands in palladium C-C coupling catalysis.
Chem. Commun. 2007, (26), 2704-2706.
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VITA
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VITA
Ziqing Lin was born in 1984 in Shanghai, China. In the summer of 2003, he was
admitted to Tsinghua University in Beijing, China, and majored in Chemistry. There, he
obtained a solid knowledge and training in analytical, inorganic, organic, and physical
chemistry. As an undergraduate junior, Ziqing had an undergraduate research program
with Prof. Xinrong Zhang in Key Laboratory for Atomic and Molecular Nanosciences of
the Education Ministry, Department of Chemistry, Tsinghua University. In 2006, he
joined Prof. Zhang’s lab for his senior design and encountered mass spectrometry, where
he found the field fascinating, that analytical chemistry especially mass spectrometry
could be an essential key to monitor and solve biomedical problems. In 2007, Ziqing was
admitted to Department of Chemistry, Tsinghua University for a 3-year Master’s program
after his bachelor degree. He continued to work on applications of ambient mass
spectrometry under the supervision of Prof. Zhang and developed a rapid screening
methodology for clenbuterol, an abused drug and speciation for 8 organic and inorganic
arsenic species. Besides his research as an analyst, Ziqing learned mechanical drawing,
and made compartments to modify the atmospheric pressure interface for mass
spectrometers as well as helping his colleagues design instrumentational devices. He also
drew an amount of cover pictures for scientific journals. Therefore, Ziqing gradually
grew into a combination of scientist, engineer, and artist. In 2010 after obtaining his
Master’s degree in analytical chemistry, Ziqing decided to come to the United States and
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135
pursue his PhD degree in Weldon School of Biomedical Engineering at Purdue
University. He joined Prof. Zheng Ouyang’s group and further worked on
instrumentation and application of mass spectrometry for gas-phase biomolecular ion
reactions, which was a joint program of both science and engineering. After building his
own mass spectrometry system, he discovered for the first time a cyclopropenylidene
reaction with protonated alkyl amines, amino acids and peptides. Ziqing also collaborated
on the development of long-distance ion-transferring tube and other ambient mass
spectrometry applications.
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PUBLICATIONS
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PUBLICATIONS
Journals
1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Gas-phase
reactions of C3H2 with protonated alkyl amines, J. Am. Chem. Soc. In preparation.
2. Lin, Z.; Tan, L.; Garimella, S.; Li, L.; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z.
Characterization of a DAPI-RIT-DAPI system for gas-phase ion/molecule and ion/ion
reactions, J. Am. Soc. Mass Spectrom. 2014, 25, 48-56.
3. Chen, C.; Lin, Z.; Garimella, S.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z.
Development of a mass spectrometry sampling probe for endoscopic analysis, Anal.
Chem. 2014, 85, 11843-11850.
4. Lin, Z.; Zhao, M.; Zhang, S.; Yang, C.; Zhang, X. In situ arsenic speciation on solid
surfaces by desorption electrospray ionization tandem mass spectrometry, Analyst
2010, 135, 1268-1275.
5. Liu, Y. ; Ma, X.; Lin, Z.; He, M.; Han, G.; Yang, C.; Xing, Z.; Zhang, S.; Zhang, X.
Imaging mass spectrometry with a low-temperature plasma probe for the analysis of
works of art, Angew. Chem.-Int. Edit. 2010, 49, 4435-4437
6. Liu, Y.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Rapid screening of active ingredients
in drugs by mass spectrometry with low-temperature plasma probe, Anal. Bioanal.
Chem. 2009, 395, 591-599.
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7. Ma, X.; Zhang, S.; Lin, Z.; Liu, Y.; Xing, Z.; Yang, C.; Zhang, X. Real-time
monitoring of chemical reactions by mass spectrometry utilizing a low-temperature
plasma probe, Analyst 2009, 134, 1863-1867.
8. Lin, Z.; Zhang, S.; Zhao, M.; Yang, C.; Chen, D.; Zhang, X. Rapid screening of
clenbuterol in urine samples by desorption electrospray ionization tandem mass
spectrometry, Rapid Commun. Mass Spectrom. 2008, 22, 1882-1888.
9. Ma, X.; Zhao, M.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Versatile platform
employing desorption electrospray ionization mass spectrometry for high-throughput
analysis, Anal. Chem. 2008, 80, 6131-6136.
Conference Oral and Poster Presentations
1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Reactions of
Biomolecule Ions with Pyrolysis-formed Carbene, Poster TP353, 62nd ASMS Annual
Conference on Mass Spectrometry and Allied Topics, Baltimore, MD, Jun 17th, 2014.
2. Lin, Z.; Tan, L.; Ouyang, Z.; Xia, Y. Instrumentation and studies for gas-phase
ion/radical reactions, Poster, 26th ASMS Sanibel Conference on Mass Spectrometry,
Clearwater Beach, Fl, US, Jan 30th-Feb 2nd, 2014.
3. Lin, Z.; Tan, L.; Chen, T.; Li, L.; Xia, Y.; Ouyang, Z. Development of a mass
spectrometer for gas phase ion/radical reactions, Oral TOA1450, 61st ASMS Annual
Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, US, Jun 11th,
2013.
4. Lin, Z.; Garimella, S.; Li, L; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z. A DAPI-RIT-
DAPI system for in-trap gas phase reactions, Oral TOC850, 60th ASMS Annual
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Conference on Mass Spectrometry and Allied Topics, Vancouver, BC, Canada, May
22nd, 2012.
5. Chen, C.; Lin, Z.; Garimella, S.; Cooks, R. G.; Ouyang, Z. Development of an
endoscopic DESI sampling probe, Poster TP034, 59th ASMS Annual Conference on
Mass Spectrometry and Allied Topics, Denver, CO, Jun 7th, 2011.