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Ion fragmentation of smallmolecules in mass

spectrometryJeevan Prasain

jprasain@uab.edu

6-2612

Feb 3, 2012

mailto:jprasain@uab.edumailto:jprasain@uab.edu

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Nomenclature: the main names andacronyms used in mass spectrometry

Molecular ion: Ion formed by addition or the removal of oneor several electrons to or from the sample molecules-Electron Impact (EI-MS). M + e- M+ + 2e-

Adduct Ion: Ion formed through interaction of two speciesand containing all the atoms of one of them plus one orseveral atoms of them (e.g. alkali, ammonium).

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Feb 3, 2012 Nielsen et al., J Nat Prod. 2011

Adduct formation in +/ve ion modes

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Contd.. Pseudomolecular ion: Ion originating from the analyte

molecule by abstraction of a proton [M-H]- or addition ofproton [M+H]+

Tandem mass spectrometry (Cooks, 1976): MS/MS (McLafferty,1978), tandem in space or time

Precursor ion/parent ion: Ions undergoing fragmentation.

Product ion/daughter ion: Ions resulting from parent/precursor

ions. Neutral loss: Fragments lost as neutral molecules

In positive ionization mode, a trace of formic acid is often added to

aid protonation of the sample molecules; in negative ionizationmode a trace of ammonia solution or a volatile amine is added to aiddeprotonation of the sample molecules. Proteins and peptides areusually analysed under positive ionization conditions andpolyphenols and acids under negative ionization conditions. In allcases, the m/zscale must be calibrated.

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Terminology..

Ionspray denotes pneumatically assisted ESIoperating at aflow rate of approximately 5 to 50uL/min.

Turboionspray is ionspray with additional heatedgas for flow rate of 0.1 to 2 ml/min.

Heated nebulizer is the trade mark of AB Sciex forAPCI.

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Precursor ion

or parent ion

Activated ion

Fragmenting ion

Neutral loss

Product ions

Schematic of CID fragmentation

What is Collision Induced Dissociation (CID)or Collisionally Activated Dissociation (CAD) ?

Other activation processes:PSD (post source-decay)ECD (electron capture dissociation)

SID (surface-induced dissociation

o

o

o

o

o

o

o

o

o

oo

oo

Collision gas

Collision cell

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Various types of MS/MS experiments

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Applications of MS/MS

Pharmaceuticals- Identification andquantification of drug metabolites, PK/PD

Academic/biotechnology- analysis ofprotein/peptides, authentification and profilingof chemical components in a crude mixture,

substructure analysis of unknowncomponents

Clinical- eg. neonatal screening, steroids in

athletes etc. Environment- eg. dioxins in fish.. Geological- eg. oil compositions

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Interpreting MS/MS spectra

Likely sites of protonation or deprotonation.

Likely leaving group.

Literature study

Fragmentation always follows the basicrules of chemistry

Where are the sites of

deprotonation/protonation?What is the most likelyleaving group in thismolecule?

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O

OH

HO

O

OHO

HO

CH2OHOH

O

OH

O

O

O

OHOH

CH2OH

OH

Puerarinm/z415

O- and C-glucosides fragmentdifferently in ESI-MS/MS

Prasain et al. J. Agric. Food Chem. 2003

220 280 300 320 360 380 m/z0

%

0

100

%

255.050

256.057

297.043

267.037

268.041281.051

307.065321.046

363.046335.061

351.044381.055

100

240 260 340

[A]

[B]

-162 Da

Yo+

-120 Da

Daidzin m/z415

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Possible product ions of puerarin m/z 415 inMS/MS

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Ion fragmentation foridentification of phase II drug

metabolites (glucuronide/sulfateconjugates)

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Wh t f t i h t i ti

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100 200 300 400

m/z

100

50

0

RelativeIntensity(%)

5985

113

133 181 224

269

Neutral loss (176 Da)

O

OHHO

OH

COOH

445

Product ion spectrum of genistein glucuronide in ESI-MS/MS

Glucosides/glucuronides conjugates are easilycleaved off by higher potential at orifice

Characteristic fragments

Aglycone

genistein

Parention

What fragment ions are characteristics

for glucuronide conjugates?

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The loss of 80 Da from the parent ion and the presence ofm/z80 in the product ion spectra are the indicative ofsulfate conjugates of like daidzein [A] and equol [B]

20 60 100 140 180 220 260 300

5.0e5

1.5e6

2.5e6

3.5e6

4.5e6

5.5e6

Intensity,cps

116.9

253.0

134.9

252.0132.991.0224.0

207.9

80.0

96.9 225.0197.0160.0

[A]

m/z, amu

20 60 100 140 180 220 260 300 340

2.0e6

4.0e6

6.0e6

8.0e6

1.0e7

1.2e7

1.4e7

Intensity,cps

121.0

119.0

135.0

79.9

93.0 241.0147.0

91.0 320.9

[B]

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Wh t h ith li h ti lf t i MS/MS?

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What happens with aliphatic sulfates in MS/MS?

Aliphatic and aromatic sulfate conjugates behave differentlyin MS/MS, aliphatic typically show m/z97 (HSO4-) and m/z80(SO3-.) Feb 3, 2012

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Source: Weidolf et al. Biomed. and Environ. Mass Spec. 15, 283-289, 198

The absence of the m/z97 fragment with the basepeak m/z80 makes the distinction between

aromatic and aliphatic sulfates

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Ch ang e in m ass

Met h y la t ion 1 4

Dem et h y lat ion - 1 4

Hy d r ox y la t ion 1 6

Acet y lat ion 4 2

Epox idat ion 1 6

Desu l f u r at ion - 3 2

Decar b ox y lat ion - 4 4

Hy d r at ion 1 8

Deh y d r at ion - 1 8

Met abo l i c r x n

Change in mass is associated with possible

metabolic reaction

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I on izat ion m ode Scan

Glu cu r on ides p os/ n eg NL 1 7 6 am u

Hex ose su gar p os/ n eg NL 1 6 2 am u

Pen t ose su g ar p os/ n eg NL 1 3 2 am uPh en o l ic su lph at e p os NL 8 0 am u

Ph osph at e n eg Pr ecu r so r o f m / z 7 9

Ar y l - GSH p os NL 2 7 5 am uAl iph at ic- GSH p os NL 1 2 9

t au r in es Pos Pr ecu r so r o f m / z 1 2 6

N-ace ty lcys te ins n eg N L 1 2 9 a m u

NL = neu t r a l l o ss.

Con juga te

Characteristic fragmentation of drugconjugates by MS/MS

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Kostiainen et al., 2003

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20 60 100 140 180 220 260

5.0e5

1.5e6

2.5e6

3.5e6

4.5e6

5.5e6

6.5e6

In

tensity,cps

107.1

135.1

159.0

133.0147.1

109.1

173.1

121.2

145.1

93.181.1

Analysis of steroids by MS/MS

20 40 80 120 160 200 2405.0e5

2.5e6

4.5e6

6.5e6

8.4e6

Intensity,cps

133.1

159.1

157.1

197.1

107.1 145.181.1 213.1 253.2183.0131.1 198.193.1 121.2

171.1

141.1105.1 179.1147.179.1

Estradiolm/z 273

Estrone m/z 271

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Estradiol Standard Curve 0.05 25 Mr = 0.9959

U ntitled 1 (Estradiol 273.2 / 107.1): "Quadratic" Regression ("1 / (x * x)" we ighting ): y = -215 x^2 + 1.7e+0 04 x + -104 (r = 0.99 59)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Concentration, u M

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

Sensitivity is an issue in quantification of steroids

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Derivatization of estradiol with dansyl chloride

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Derivatization of estradiol with dansyl chlorideleads to the formation of E2-dansyl (m/z506)

Source: Nelson et al. Clinical Chemistry, 2004

Easyprotonation

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D i i i d l h l i

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Time, min

1 3 5 7 90

40

100

160

220

280

340

400

460

Intensity,cps

5.62

5.80

6.26

6.62 7.807.095.50 7.53 8.005.228.494.88

4.37 8.63

MRM chromatogram (m/z506/171) 50 picomoledansylated E2

Derivatization tremendously helps increasesensitivity of E2

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Calibration curve for dansylated E2 showinglinearity from 0.005-100 nM concentration range

(r = 0.999)

0 10 20 30 40 50 60 70 80 90 100

Concentration, nM

5.0e5

2.0e6

3.5e6

5.0e6

6.5e6

8.0e6

Area,counts

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Identification of Natural productsBy MS/MS

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Neutral losses (162 and 180) is useful in deciding

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m/z

100

50

0

100 200 300 400

m/z

100

50

0.0

Rel.

Int.(%)

253

109

161

224

253

271

433

433

180 Da

162 Da

Neutral losses (162 and 180) is useful in decidingwhether sugar is attached to an aromatic ring or not

O

O-

HO

O

O

OHO

OH

HO

CH2OH

O

O-

O

O

OH

OHO

HO

HO CH2OH

162 Da

Possible

structure

Hexose attachedTo the ring B

162 loss is an indicativeof aromatic attachment

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I lik i i d i i dil

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m/z

100

50

0

50 100 150 200 250

m/z

100

50

0

R

el.Int.(%) 65 107

117

151

225

269

63

91107

133

159 180196

224

240

269

Genistein

Apigenin

149

Isomers like genistein and apigenin are readilyseparated by tandem mass spectrometry

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C i f d t i h l

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Comparison of product ions helpelucidate the unknown structures

150 200 250 300 350 400

100

%

255268

282311

-120431

Monohydroxylatedpuerarin

200 250 300 350 400

100

50

0

267 295

\

415

-120

Puerarin

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Substructure analysis in ESI-MS/MS(dereplication and partial identification

of natural products)

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Fragmentation of basic taxoids from T

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Fragmentation of basic taxoids from T.Wallichianaextract

Prasain et al. Anal Chem, 2001

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ESI MS/MS spectra of taxoids (1 3) Peaks m/z 194

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ESI-MS/MS spectra of taxoids (1-3). Peaks m/z194and 210 represent the intact alkaloid side chain.

AlkaloidSide chainm/z 210

DiterpenoidScaffold

Loss of 60 or42

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MS/MS precursor-scan spectra of typical alkaloid side chains

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MS/MS precursor-scan spectra of typical alkaloid side chainsto identify the basic taxoids compounds in an ethyl acetate

extract of T. wallichiana.

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600 620 640 660 680 700 720 740 760 780 800 820 840 860 880

605

622

714

742

758 800

612 628 668 710

742

806.2 848 862

668684

726

744

758

770 786

686

710

728

744

770786

800

A

B

C

D

Scaffold (m/z 309)

Side chain (m/z 210)

Side chain (m/z 252)

Side chain (m/z 194)

m/z

Intensity

Comparison of precursor scan spectra obtained from thescaffold m/z309 and side chain m/z194, 210 and 252

Taxoids with scaffold m/z309 and alkaloid side

chains are shown by dashed linesFeb 3, 2012

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References

1. Electrospray Ionization Mass Spectrometry by Richard B.Cole.

2. Stefanowicz P, Prasain JK, Yeboah KF, Konishi Y. Detectionand partial structure elucidation of basic taxoids fromTaxus wallichianaby electrospray ionization tandem massspectrometry. Anal Chem. 2001;73:3583-9.

3. Prasain J.K., Wang C.-C., Barnes S. Mass spectrometricanalysis of flavonoids in biological samples. Free RadicalBiology & Medicine, 37: 1324-1350, 2004.

4. William Griffiths. Tandem mass spectrometry in the study of

fatty acids, bile acids and steroids. Mass SpectrometryReviews, 2003;22:81-152.

Feb 3, 2012

http://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/15454273?ordinalpos=16&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum