1 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 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? O O O HO O HO HO HO OH O O HO HO OH O-
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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
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?
O
O O
HO
O
HO
HOHO
OH
O
OHO
HOOH
O-
2
Isotopic pattern and intensity of ions indicates the number of carbons and hetero atoms
Ion fragmentation for identification of phase II drug
metabolites (glucuronide/sulfate conjugates)
3
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
What fragment ions are characteristics for glucuronide conjugates?
m/z 445
-176 Da
60 120 180 240 300
Mass/Charge, Da
0
500113.0248
107.0507
151.0399
283.082585.0313
117.020359.0175175.0244
87.0108
327.0740129.0197
231.0868187.0977327.1434
265.1245
O
O
OH
OO
-O
OH
OH
OH
HO
O
O
-43.99 DaCO2
-43.99 DaCO2
Calc. m/z 327.072Calc. m/z 151.040
Rel
ativ
e in
tens
ity
The neutral loss of 176 Da is an indicative of glucuronide metabolites
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MSMS of m/z 429 indicate that it may be daidzein glucuronide
m/z 253
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
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Mass/Charge, Da
60 80 100 120 140 160 180 200
700 91.0552
76.0402
194.0825
148.0756 176.0691 194.1893120.080890.782979.0543
65.0408185.0633166.1322
Re
lativ
e in
ten
sity
Calc. m/z194.081
Among the annotated list of compounds by Metlin - phenylacetylglycine’s validation by
MS/MS interpretation
A medium chain dicarboxylic fatty acid with m/z 241.109 [M-H]-
3200
60 90 120 150 180 210 240
197.1193
241.1092
242.1153179.1091153.1274
69.0382 125.0979 223.1002
-18.00 da
-43.9899 daCO2
-62 da44+18
-43.991 daCO2
Calc. 43.9898
Re
lativ
e in
ten
sity
m/z
C12H17O5 [M-H]-
Calc. m/z 241.1081
Many metabolites, unidentified by the Metlin database
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
Stefanowicz et al. Anal Chem, 2001
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Conclusions
• Identifying unknown metabolites is a significant analytical challenge in metabolomics and it requires integrated analytical and bio-informative approaches.
• The use of high-resolution MS and MSn provides important information to propose structures of fragment and precursor ions.
• Only an integrated approach can describes multitude of metabolites present in a biological sample.