ion fragmentation of small molecules in mass spectrometry · msms of m/z 429 indicate that it may...
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Nomenclature: the main names and acronyms used in mass spectrometry
• Molecular ion: Ion formed by addition or the removal of one or several electrons to or from the sample molecules-Electron Impact (EI-MS). M + e- M+• + 2e-
• Adduct Ion: Ion formed through interaction of two species and containing all the atoms of one of them plus one or several atoms of them (e.g. alkali, ammonium).
Nielsen et al., J Nat Prod. 2011
Adduct formation in +/–ve ion modes
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Molecules with inherent positive charge- molecular weight and m/z are same
Increasing metabolite coverage using +ve and –ve ion mode
Source: Nordstrom et al. Analytical Chemistry, 2007
Representative Q1 scans of a methanolic extract of human blood serum
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Contd..• Pseudomolecular ion: Ion originating from the analyte
molecule by abstraction of a proton [M-H]- or addition of proton [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 ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. Proteins and peptides are usually analysed under positive ionization conditions and polyphenols and acids under negative ionization conditions. In all cases, the m/z scale must be calibrated.
Isotopic distribution and MS
• 1H = 99.9%, 2H = 0.02%
• 12C = 98.9%, 13C = 1.1%
• 35Cl = 68.1%, 37Cl = 31.9%
• Monoisotopic mass - the mass of the most abundant isotope
• Average mass- the abundance weighted mas of all isotopic components.
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Precursor ionor 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) ?
o
o
o
o
o
o
o
o
o
oo
oo
Collision gas
Collision cell
Various types of MS/MS experiments
Hopfgartner etal. J. Mass Spectrom, 2004
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Applications of MS/MS
• Pharmaceuticals- Identification and quantification of drug metabolites, PK/PD
• Academic/biotechnology- analysis of protein/peptides, authentification and profiling of chemical components in a crude mixture, substructure analysis of unknown components
• Clinical- eg. neonatal screening, steroids in athletes etc.
• Environment- eg. dioxins in fish..• Geological- eg. oil compositions…
Interpreting MS/MS spectra
• Likely sites of protonation or deprotonation.
• Likely leaving group.
• Literature study
Fragmentation always follows the basic rules of chemistry
Where are the sites of deprotonation/protonation?What is the most likely leaving group in this molecule?
OHO
O
O
O
CH2OHHO
HOHO
OCOOH
OHOHOH
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O
OH
HO
O
OHO
HO
CH2OHOH
O
OH
O
O
O
OHOH
CH2OH
OH
Puerarinm/z 415
O- and C-glucosides fragment differently 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/z 415
Ion fragmentation for identification of phase II drug
metabolites (glucuronide/sulfate conjugates)
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100 200 300 400m/z
100
50
0
Rel
ativ
e In
ten
sity
(%
)
5985
113133 181 224
269
Product ion spectrum of genistein glucuronide in ESI-MS/MS
Glucosides/glucuronides conjugates are easily cleaved off by higher potential at orifice
genistein
What fragment ions are characteristics for glucuronide conjugates?
60 120 180 240 300Mass/Charge, Da
Inte
ns
ity
0
400 141.0192
113.0232
111.0082
85.0303
193.0353147.030059.0170
317.053889.0243
71.0153175.0252 201.9899
176.04 Da
-H
Putative identification-a glucuronide conjugate of tetrahydroxybenzene
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MSMS of m/z 429 indicate that it may be daidzein glucuronide
m/z 253
60 80 100 120 140 160 180 200 220 240 260 280
Exact mass: 258.9818
Exact mass: 179.0350
Exact mass: 135.0452
or
Caffeic acid 4- or 3- sulfate
3.1e4 179.0344
258.9909
180.0371135.0439 259.1177259.9938
179.120291.0552 241.1063197.1191178.4728
79.9465 DaSO3, exact mass 79.9568
High resolution accurate MS/MS help identify sulfated conjugates in unknowns
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What happens with aliphatic sulfates in MS/MS?
Aliphatic and aromatic sulfate conjugates behave differently in MS/MS, aliphatic typically show m/z 97 (HSO4-) and m/z 80 (SO3-.)
Source: Weidolf et al. Biomed. and Environ. Mass Spec. 1988
The absence of the m/z 97 fragment with the base peak m/z 80 makes the distinction between
aromatic and aliphatic sulfates
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Change in mass
Methylation 14Demethylation -14Hydroxylation 16Acetylation 42Epoxidation 16Desulfuration -32Decarboxylation -44Hydration 18Dehydration -18
Metabolic rxn
Change in mass is associated with possiblemetabolic reaction
Ionization mode Scan
Glucuronides pos/neg NL 176 amuHexose sugar pos/neg NL 162 amuPentose sugar pos/neg NL 132 amuPhenolic sulphate pos NL 80 amuPhosphate neg Precursor of m/z 79 Aryl-GSH pos NL 275 amuAliphatic-GSH pos NL 129taurines Pos Precursor of m/z 126N-acetylcysteins neg NL 129 amu
NL = neutral loss.
Conjugate
Characteristic fragmentation of drug conjugates by MS/MS
Kostiainen et al., 2003
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20 60 100 140 180 220 2605.0e5
1.5e6
2.5e6
3.5e6
4.5e6
5.5e6
6.5e6
Inten
sity, cps
107.1
135.1
159.0
133.0147.1
109.1
173.1
121.2
145.193.181.1
Analysis of steroids by MS/MS
+OH2
HO
HO
+
m/z 159
+HO
m/z 107
20 40 80 120 160 200 2405.0e5
2.5e6
4.5e6
6.5e6
8.4e6
Intensity, cps
133.1
159.1
157.1
197.1107.1 145.1
81.1 213.1 253.2183.0131.1 198.193.1 121.2171.1
141.1105.1 179.1147.179.1
HO
O
OH+
+OH 2
m/z 135
Estradiolm/z 273
Estrone m/z 271
Estradiol Standard Curve 0.05 – 25 µMr = 0.9959
U ntitled 1 (Estrad io l 273 .2 / 107 .1) : "Quadra tic" Regression ("1 / (x * x)" we igh ting ): y = -215 x^2 + 1 .7e+004 x + -104 ( r = 0.9959)
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 25C oncentration , uM
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 chlorideleads to the formation of E2-dansyl (m/z 506)
Source: Nelson et al. Clinical Chemistry, 2004
Easy protonation
Time, min
1 3 5 7 90
40
100
160
220
280
340
400
460
Intensity, cps
5.62
5.806.26
6.62 7.807.095.50 7.53 8.005.228.494.88
4.37 8.63
MRM chromatogram (m/z 506/171) 50 picomoledansylated E2
Derivatization tremendously helps increase sensitivity of E2
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Calibration curve for dansylated E2 showing linearity 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
Substructure analysis in ESI-MS/MS(dereplication and partial identification
of natural products)
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Fragmentation of basic taxoids from T.Wallichiana extract
Prasain et al. Anal Chem, 2001
ESI-MS/MS spectra of taxoids (1-3). Peaks m/z 194 and 210 represent the intact alkaloid side chain.
AlkaloidSide chainm/z 210
Diterpenoid Scaffold
Loss of 60 or42
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MS/MS precursor-scan spectra of typical alkaloid side chains to identify the basic taxoids compounds in an ethyl acetate
extract of T. wallichiana.
600 620 640 660 680 700 720 740 760 780 800 820 840 860 880
605622
714
742
758 800
612 628 668 710
742
806.2 848 862
668684
726
744
758770 786
686710
728
744770
786
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
Inte
nsi
ty
Comparison of precursor scan spectra obtained from thescaffold m/z 309 and side chain m/z 194, 210 and 252
Taxoids with scaffold m/z 309 and alkaloid side chains are shown by dashed lines
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References
1. Electrospray Ionization Mass Spectrometry by Richard B. Cole.
2. Stefanowicz P, Prasain JK, Yeboah KF, Konishi Y. Detection and partial structure elucidation of basic taxoids from Taxus wallichiana by electrospray ionization tandem mass spectrometry. Anal Chem. 2001;73:3583-9.
3. Prasain J.K., Wang C.-C., Barnes S. Mass spectrometric analysis of flavonoids in biological samples. Free Radical Biology & Medicine, 37: 1324-1350, 2004.
4. William Griffiths. Tandem mass spectrometry in the study of fatty acids, bile acids and steroids. Mass Spectrometry Reviews, 2003;22:81-152.
5. Yi et al., Anal Bioanal Chem. 2006.