Special thanks to: Jim Scrivens, Roman Zubarev, Claudia Blindauer, Ann Dixon, Scott Mcluckey, Fred W. McLafferty, Frank Turecek, Ron Heeren, and many others for helpful slides.

Also thanks to my Warwick research group: Anna Pashkova, Andrea Clavijo-Lopez, Pilar Perez-Hurtado, Rebecca Wills, Huilin Li, Terry Lin, Yulin Qi, Andrew Soulby for help with the course.

CH908: Mass Spectrometry

Spring 2011

Professor Peter B. O’Connor

15, 1 hour lectures- slides will be posted online prior to the lecture.

10-15 problem sets, about one for each lecture – these will not be marked- problem sets will be posted online prior to the lecture. - solutions will be posted online as well, sometime thereafter.

4 workshops for helping with the problem sets – expected to start with a lecture interpreting an example spectrum.

2 marked homework sets (25% of final mark, each)

1 Exam. Exam questions will be derived from the problem sets. (50% of final mark)

Fred W. McLafferty and Frantisek Turecek (1993), Interpretation of Mass Spectra – Fourth Edition, University Science Books, ISBN 0-935702-25-3 (Electron Impact spectra) For this lecture, read chapters 2-3.

Edmond de Hoffmann and Vincent Stroobant (1999), Mass Spectrometry: Principles and Applications 2nd Edition, Wiley, ISBN 0-471-48566-7 (General text)

Richard B.Cole (1997), Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications, Wiley, ISBN 0-471-14564-5 (ESI / Instrumentation / Applications / LC-MS)

Objectives for this lecture:

• Introduce the following concepts:– masses of elements and molecules– isotopes and isotope distributions– mass accuracy and its limitations– mass resolving power– Electron Impact (odd electron ions)– Chemical Ionization (even electron ions)– Tandem mass spectrometry

Essentials of a Mass Spectrometer Sample inlet system (possibly including

chromatography) Ion source Mass analyzer (determines mass range,

accuracy, and resolving power) Ion detection system Data system controls instrument and usually has

tools to assist processing data Connection to on-line database in some cases (-

omics)

IonSource

Mass Analyzer

IonDetector

Inlet DataSystem

VacuumPumps

SampleIntroduction

DataOutput

Mass Spectrometer

What does a Mass What does a Mass Spectrometer Do?Spectrometer Do? Generates ions (positive or negative) from

samples introduced directly or from a GC or HPLC Separates ions according to their mass-to-charge

ratios, m/z. Often, z=1, but electrospray ionisation gives multiply charged ions.

Collects ions at each m/z and records relative abundances

A data system then can be used to process the recorded data (normalisation, background subtraction, mass range displayed, etc.)

Plots normalised relative abundance against m/z - this is the mass spectrum.

Advantages of Mass Advantages of Mass SpectrometrySpectrometry NEAR UNIVERSAL APPLICABILITY

Almost all substances give a mass spectrum SELECTIVITY

High Resolving Power allows selection of one component from a complex mixture

SPECIFICITY Exact molecular weight is often specific to the

compound under investigation; observation of a chosen fragmentation in MS often identifies a given component in a mixture

SENSITIVITY Detection levels as low as 1 femtomole (10-15

moles). Complete structure from less than 100 femtomoles

SPEED

Limitations of Mass Limitations of Mass SpectrometrySpectrometryCan sometimes have difficulty in

distinguishing isomers from each other. It can distinguish isobaric compounds, e.g.

CO and N2, only if the resolving power is sufficiently high

May decompose or isomerize compounds during ionisation process. Secondary or higher structure observed in solution may be lost during ionisation process

Usually needs very, very, very pure samples – so sample preparation is THE KEY to obtaining good spectra.

Limitations of Mass Spectrometry

Detailed mechanism of ionisation and fragmentation processes are not fully understood; sometimes difficult to predict the mass spectrum of a particular molecule from first principles

Getting detailed structural information from the spectra requires a solid understanding of fragmentation mechanisms.

Quantitation requires use of an internal standard such as a deuterated analog

1. Gravitational mass. Newton’s law of gravitation:

Fg = GmM/r2 - gravitational force

G – gravitational constant;m, M – masses of spherical bodies;r – center-of-mass distance.

2. Inertial mass. Newton’s second law of mechanics:

Fi = mdv/dt, - external force

v - velocity.

Mass equivalency principle. Gravitational and inertial masses are

equivalent. Confirmed experimentally to the accuracy of 10-12 (1971).

Mass and energy equivalency. Einstein’s law:

E = mc2, - total energy

c – speed of light, 3108 m/s.

Example: 1 eV = 1.0710-9 u (Da).

rm

M

What is mass?

Who Was John Dalton?

John Dalton (1766-1844) of Manchester, England was the first to discover the empirical Law of Multiple Proportions (around 1804): When any two elements are observed to form more than one compound between them, the mass ratios in one compound will be related to the mass ratios in the other in the proportions of whole numbers. Since hydrogen was the lightest element known, Dalton assumed that hydrogen should have an atomic mass of 1.

Source: www.daltonics.bruker.com/about/johndalton.htm

Mass Units

Since the IUPAC meeting of 1968, the Dalton (Da) is defined as 1/12th of the mass of the 12C isotope of carbon.

Note: the atomic mass unit (amu) was a previous unit based on 1/16th of the mass of 16O – it’s usage was officially discontinued in 1968, but it lingers in the literature causing confusion.

Mass: M = Σmene,Isotope Mass Abundance Chemical Deviation from the mass whole number

1H 1.00782510 99.9852% 1.00794 +0.00792H (D) 2.01410222 0.0148%

12C 12.0(0) 98.892% 12.011 +0.01113C 13.0033544 1.108%

14N 14.00307439 99.635% 14.00674 +0.00715N 15.0001077 0.365%

16O 15.99491502 99.759% 15.9994 -0.000617O 16.9991329 0.037%18O 17.99916002 0.204%

31P 30.9737647 100% 30.9737647 -0.0262

32S 31.9720737 95.0% 32.066 +0.06633S 32.9714619 0.76%34S 33.9678646 4.22%36S 35.967090 0.014%

What is Molecular Mass?

What is a mass spectrum?

“A+1” elements(carbon, nitrogen, hydrogen)

(fluorine, phosphorus, cesium, sodium, iodine)

Monoisotopic “A” elements

“A+2” elements(oxygen, chlorine, bromine, silicon, sulfur)

Elemental Compositions of Metals

Magnesium

23.98504 78.724.98584 1025.98259 11.3

Aluminum

26.98153 100

Titanium

45.95263 846.9518 7

47.94795 7448.94787 5.549.9448 5

Iron

53.9396 5.855.9349 92

Zinc

63.9291 4965.926 28

66.9271 467.9249 18.6

Selenium

75.9192 976.9199 7.677.9173 23.579.9165 49.881.9167 9

Palladium

101.9049 1103.9036 11104.9046 22105.9032 27107.903 28

109.9045 12silver

106.9041 52108.9047 48

Gold

196.9666 100

Mercury

197.9668 10198.9683 17199.9683 23200.9703 13201.9706 30203.9735 7

Lead

203.973 1.5205.9745 23206.9759 22.6207.9766 52.3

Uranium

235.0439 0.7238.0508 99.3

CRC Handbook of Chemistry and Physics, 48th Edition, 1967

• monoisotopic mass dominates up to MW ~1100 - 1500

• above MW ~7000, the monoisotopic peak is vanishingly small

• becomes more symmetric

• the width grows sublinearly. For <3 kDa, MW/FWHM ~1100, for 10 kDa, MW/FWHM ~2000

• the most abundant mass is 0 to 1 Da below the average mass

• fine structure for all peaks but monoisotopic.Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C.

Anal. Chem. 1983, 55, 353-356.

MW 1000 MW 2000 MW 3000 MW 4000

FWHM

How do isotopic distributions change with mass?

Nominal mass: me is the integer mass value for the most abundant isotope (H=1, etc.).

Monoisotopic mass: me is the exact mass value for the most abundant isotope (H=1.00782510, etc.).

Average mass: me is the chemical (average) atomic mass value (H=1.00794, etc.).

Isotopic cluster (distribution): a group of isotopic peaks representing the same molecule.

Most abundant mass: such in the isotopic cluster.

Mass quantities:Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C.

Anal. Chem. 1983, 55, 353-356.

Molecular mass is the isotopic distribution!

How to calculate the isotopic distribution?iNi pp

iNi

NiP

)1(

)!(!

!)(

N= number of atomsi = ith isotopep = probability of being heavy isotope (e.g. 13C)

Note: the total isotopic distribution is the convolution of the individual isotopic distributions for each possible isotope.

Yergey, J. A. Int. J. Mass Spectrom. Ion Physics, 1983, 52, 337-349. Rockwood, A. L.; Van Orden, S. L.; Smith, R. D. Anal. Chem. 1995, 67, 2699-2704.

Yergey, J. A. Int. J. Mass Spectrom. Ion Physics, 1983, 52, 337-349. Rockwood, A. L.; Van Orden, S. L.; Smith, R. D. Anal. Chem. 1995, 67, 2699-2704.

Fine structure of isotopic peaks

RP = 300k

RP = 600k

RP = 1500k

RP = 2.7M

Is the Average Mass Reliable?

Inherent uncertainty of average mass is ca. 10 ppm.

Is average mass reliable?

Underestimation by 0.45±0.10 Da.

Zubarev RA, Demirev PA, Håkansson P, Sundqvist BUR. Anal. Chem. 1995, 67, 3793-3798.

0.1 Da!Minimal

U nre gist ered

12 .37 012 .36 512. 36012. 3 5512. 350

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

"Averagine" concept :

C4.9384

H7.7583

N1.3577

O1.4773

S0.0417

mmono

"Averagine" - average amino acid residue

maverage

= 111.1254 Da

= 1000/ 9.000 Da

Senko MW, Beu SC, McLafferty FW, JASMS, 1995, 6, 229-233.

Effect of poor statistics

Hundreds to thousands of ions are needed to identify reliablythe monoisotopic mass of a peptide with MW >1 kDa!

Mmono

TheoryMmono

Experiment

Statistical scatter in isotopic abundances

Kaur, P.; O'Connor, P. B. Use of statistical methods for quantitative determination of the number of trapped ions Anal Chem 2003, 76, 2756-2762.

100 ions 1000 ions

10000 ions∞ ions

100 ions300 scans

5000 ions300 scans

Myoglobin

C60

Peak position determination

0

1000

2000

3000

4000

5000

6000

1010 1011 1012 1013 1014 1015 1016 1017m/z

0

1000

2000

3000

4000

5000

6000

1010 1011 1012 1013 1014 1015 1016 1017m/z

Constant noise

Noise NoisePeak

Noise level(background, baseline)

Peakintensity

Peak-to-peaknoise

Apex(maximum)

FWHM

Peak pedestal

Peakcentroid

Peak area

Peak position determination (Centroiding)

2500

3000

3500

4000

4500

5000

5500

1012 1013 1013 1013 1013 1013 1014

Apex fitting

1012.989+/-0.025

0

1000

2000

3000

4000

5000

6000

1010 1011 1012 1013 1014 1015 1016 1017m/z

y = m4+m3*exp(-(M0-m1)^2/m2)

ErrorValue

0.00975291012.9958m1

0.015132930.37741281m2

54.995453045.6712m3

13.863121586.3965m4

NA49869069Chisq

NA0.95045653R

Peak position:1012.996 +/- 0.010

Curve fitting

1012.996+/-0.010

ii

ii

I

iC

zmI )/(1012.989 ± 0.012

Center of mass determinationError = FWHM/k

k = f(Statistics, S/N)

Known: there are ☻and ☻inside.☻? ☻?

Extracting Information from Mass

A priori:M(empty box) = 1.0000 gM(☻) = 3.141593 gM(☻) = 2.718282 gMeasured: M(box) = 10.000±0.002 g

BlackBox

☻ ☻ ☻☻ ☻1 1 5.8601 2 8.5781 3 11.2962 0 6.2832 1 9.0022 2 11.7202 3 14.4383 0 9.4253 1 12.1433 2 14.861

Using “Exact Mass” for Elemental Composition

Suppose you have a peak at 128.0454? What elemental compositions are possible for such a peak?

Limits of Mass Accuracy

Dougherty RC, Marshall AG, Eyler JR, Richardson DE, Smaller REJASMS, 1994,5, 120-123.

Current mass standard: based on 12C:

12Csolid 12Cgas 12C+ + e-

7.42 eV 11.26 eV

18.7 eV = 20.7 nDa = 1.7 ppb for 12C

Resolving power and peak separation

0

0.2

0.4

0.6

0.8

1

999.00 1000.0 1001.0 1002.0m/z

R=3000

m/z

FWHM: R = (m/z)/( m/z)1.0000

0

0.2

0.4

0.6

0.8

1

999.00 1000.0 1001.0 1002.0m/z

R=2000 0.9995

0

0.2

0.4

0.6

0.8

1

999.00 1000.0 1001.0 1002.0m/z

R=1400 0.9885

0

0.2

0.4

0.6

0.8

1

999.00 1000.0 1001.0 1002.0m/z

R=1000

0.706

R = (m/z)0/FWHM(m/z)FWHM: full width at half maximum

For isotopic resolution at MW = MW0, one needs RFWHM>1.4MW0

FWHM

Resolving power in FTMS

1328 1328 1329 1329 1330 1331 1331 1332 1332m/z

Benefits of High Resolution

MALDI FTMSR > 500,000

K/ Q substitution36.4 mDa

Q

Q

Q

Q

K

K

K

K

Instrumentation: 4.7 Tesla FTMS

Close-mass separation in FTMS

FourierTransformMass Spectrometry

Shi SD-H, Hendrickson CL, Marshall AG,PNAS 1998, 95, 11532-11537.

Close-mass separation in FTMS

Close mass separation in FTMS

Shi SD-H, Hendrickson CL, Marshall AG,PNAS 1998, 95, 11532-11537.

Electron Impact : Mass Electron Impact : Mass spectrometry of volatile spectrometry of volatile materialsmaterials Ionization by electron impact (EI) Radical ion formed, thus... Significant fragmentation Can use libraries (250K compounds) or interpret

spectra Very commonly used with gas chromatography Chemical ionisation (CI) for softer ionization Limited to volatile, stable compounds < 1000 Da

IonSource

Mass Analyzer

IonDetector

Inlet DataSystem

VacuumPumps

SampleIntroduction

DataOutput

Mass Spectrometer

194

67 109

5582

42

16513694

40 60 80 100 120 140 160 180 200

Abundance

Mass (amu)

Mass Spectrum

NC

CNH

CO

C

O

N

N

C H

C 3H

C3H

MassSpectrometer

Typical sample: isolatedcompound (~1 nanogram)

Mass Spectrometry

Electron Impact

M + eM + e-- → M→ M+.+. + 2e + 2e--

Many Fragments….

Electron impact ionisation (EI)The ionization potential is the electron

energy that will produce a molecular ion. The appearance potential for a given fragment ion is the electron energy that will produce that fragment ion.

Most mass spectrometers use electrons with an energy of 70 electron volts (eV) for EI.

This is (usually) the most sensitive and stable value.

Decreasing the electron energy can reduce fragmentation, but it also reduces the number of ions formed.

~70 Volts

+

_

+

_

e- e-e-

++ ++++

_

Electron Collector (Trap)

Repeller

ExtractionPlate

Filament

toAnalyzer

Inlet

Electrons

NeutralMolecules

PositiveIons

Electron Impact ionisation source

Electron impact ionisation (EI) Sample introduction

Heated batch inlet Heated direct insertion probe Gas chromatography Liquid chromatography (particle-beam interface)

Benefits Well-understood Can be applied to virtually all volatile compounds Very reproducible mass spectra Fragmentation provides structural information Libraries of mass spectra can be searched for EI mass spectral

"fingerprint" Limitations

Sample must be thermally volatile and stable The molecular ion may be weak or absent for many

compounds.

Chemical ionisationMethane:

CH4 + e -----> CH4+. + 2e ------> CH3

+ + H.

CH4+. + CH4 -----> CH5

+ +CH3.

CH4+. + CH4 -----> C2H5

+ + H2 + H.

Isobutane:

i-C4H10 + e -----> i-C4H10+. + 2e

i-C4H10+. + i-C4H10 ------> i-C4H9

+ + C4H9 +H2

Ammonia:

NH3 + e -----> NH3+. + 2e

NH3+. + NH3 ------> NH4

+ + NH2.

NH4+ + NH3 --------->N2H7

+

Even-Electron Ions Under EI conditions, M+. ions are formed and a

major fragmentation process is the loss of a radical, R., producing an even-electron ion.

Once a radical has been lost, all subsequent fragmentations involve the loss of a molecule to form further even-electron ions.

Under CI conditions, an even-electron ion, such as MH+, is formed; subsequent fragmentations involve the loss of a molecule to form further even-electron ions.

Sites of Protonation In order to rationalise the fragmentation of MH+

ions, one must consider at which sites in the sample molecule the proton is attached. The spectrum may then be understood in terms of the fragmentation of the different types of MH+

ions. In general, protonation occurs on heteroatoms

having lone pairs of electrons, such as O, N, and Cl. This frequently followed by elimination of a molecule containing the hetero-atom. Other common protonation sites are aromatic rings and regions of unsaturation.

Even Electron Ions

Ephedrine ionised by methane CI may protonate for example on the O atom of the OH group:

Protonation on the N atom leads to the loss of CH3NH2 by a similar mechanism, yielding an ion of m/z 135. Both m/z 148 and 135 are observed in the CI spectrum, indicating the presence of OH and HNCH3 groups in the molecule.

Ephedrine EI and CI Spectra

Ephedrine, 165 Da, gives an EI spectrum dominated by the m/z 58 fragment ion and no observable M+. ion.

Methane CI gives an MH+ ion at m/z 166 and fragments at m/z 148, 135 and 58 due to protonation on the OH and NHCH3 groups or on the aromatic ring respectively

Chemical ionisation Chemical ionization mass spectrometry is the

first so-called `soft' ionization technique, to produce information about the molecular weight in many cases where electron impact mass spectrometry fails to do so.

One reason for this difference is the fact that whereas in electron impact ionization, the energy transfer distribution may include a small fraction with energies more than 10 eV above the ionization potential, the energy transfer in chemical ionization processes other than charge exchange does not exceed 5 eV with the more energetic protonating reagents. - this value can be controlled somewhat by selection of different reagent gases.

Chemical ionisation Sample introduction

Heated batch inlet Heated direct insertion probe Gas chromatography Liquid chromatography (particle-beam interface)

Benefits Often gives molecular weight information through molecular-

like ions such as [M+H]+, Even when EI would not produce a molecular ion. Simple mass spectra, fragmentation reduced compared to EI

Limitations Sample must be thermally volatile and stable Less fragmentation than EI, fragment pattern not informative

or reproducible enough for Library search Results depend on reagent gas type, reagent gas pressure or

reaction time, and nature of sample.

Tandem Mass Spectrometry or MS/MS

MS/MSMS/MS/MS, or MS3

Benefits: 1.Extremely high specificity2.More structural information

Limitations:1.Isolation window2.Fragmentation efficiency3.Ion Losses

Self assessment• What is the exact mass (to 0.1 mDa) of

dihydroxy benzoic acid? Its M+• ion? Its M+H+ ion?

• A series of peaks spaced 2 Da apart indicate what?

• A 10 kDa protein ion has isotope peaks which are 0.01 Da wide (FWHM). What’s the resolution? What is the minimum resolution needed to separate two adjacent isotopes to the half height?

• In a mass spectrum, are fragments good?

Fini…

CH908: Mass spectrometryLecture 1

Missing: 1. Nitrogen rule slide and examples2. Even electron ion fragmentation rule3. R+DB slide and examples4. The concept of MS/MS, Msn, isolation bleedthrough, specificity improvement5. Energetics of ionization, proton affinity (CI)

Chemical ionisation Chemical ionization uses ion-molecule

reactions to produce ions from the analyte. The chemical ionization process begins when a reagent gas such as methane, isobutane, or ammonia is ionized by electron impact.

A high reagent gas pressure (or long reaction time) results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals.

Some of the products of these ion-molecule reactions can react with the analyte molecules to produce analyte ions.

Chemical ionisationA possible mechanism for ionization

in CI occurs as follows: Reagent (R) + e- → R+ + 2 e- R+ + RH → RH+ + R RH+ + Analyte (A) → AH+ + R      

In contrast to EI, an analyte is more likely to provide a molecular ion with reduced fragmentation using CI. However, similar to EI, samples must be thermally stable since vaporization within the CI source occurs through heating.

Electron impact ionisation (EI)A beam of electrons passes through

the gas-phase sample. An electron that collides with a neutral analyte molecule can knock off another electron, resulting in a positively charged ion. The ionization process can either produce a molecular ion which will have the same molecular weight and elemental composition of the starting analyte, or it can produce a fragment ion which corresponds to a smaller piece of the analyte molecule.

Schematic of electron impact source

Chemical ionisationChemical Ionization (CI) is applied to

samples similar to those analyzed by EI and is primarily used to enhance the abundance of the molecular ion.

Chemical ionization uses gas phase ion-molecule reactions within the vacuum of the mass spectrometer to produce ions from the sample molecule.

Schematic of chemical ionisation source

Chemical ionisationThe chemical ionization process is

initiated with a reagent gas such as methane, isobutane, or ammonia, which is ionized by electron impact.

High gas pressure in the ionization source results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals.

Some of the products of the ion-molecule reactions can react with the analyte molecules to produce ions.     

Chemical ionisation Another reason is the greater stability of even-

electron protonated ions (MH+) compared with radical molecular ions (M+.).

Much of the additional power of chemical ionization mass spectrometry arises from the fact that the characteristics of the CI mass spectrum produced are highly dependent on the nature of the reagent gas used to ionize the sample. As a consequence, it is possible to control the structural information observed by varying the nature of the reagent gas used.