Interpreting MS/MS spectra · 2018-07-26 · 65.0408 166.1322 185.0633 Relative intensity Calc. m/z...
Transcript of Interpreting MS/MS spectra · 2018-07-26 · 65.0408 166.1322 185.0633 Relative intensity Calc. m/z...
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
HOHO
OH
O
OHO
HOOH
O-
2
Isotopic pattern and intensity of ions indicates the number of carbons and hetero atoms
in the molecular ion
1H = 99.9%, 2H = 0.02%12C = 98.9%, 13C = 1.1%35Cl = 68.1%, 37Cl = 31.9%
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
4
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
5
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
Prasain et al., J Mass Spectrom. 2017
6
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
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
7
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
8
MS based-Lipidomics
Profiling phospholipids and sphingosinesin a complex mixture using MS/MS
PENeutral Loss scan 141
PC & SMPrecursor ion scan 184
PSNeutral Loss scan 185
9
How to profile sphingolipidsin a complex mixture using MS/MS?
m/z 264 is a characteristic ion for all compounds containing a sphingosine backbone
Phosphatidylcholine loses a methyl group to form a negatively charged, pseudomolecular ion
Phospholipids may undergo demethylation and then theloss of the fatty acyl groups from glycerophosphocholinebackbone.
PO-
O
O
N+OO
O
R1O
R2O
PO-
O
O
NO
OO
R1O
R2O [M-15]-
CID
CH3 O
O
CH3 OCH3
O
+
O
R2O
O
R1O +
10
300 360 420 480 540 600 660 700
8.7e4
Intensity, cps
649.0
623.1
651.0
605.0
631.1621.0594.8 636.8
647.1538.8394.7407.6
C22:0Calc. 622.6133
C24:1Calc. 648.6289
C20C16
-H2O
Precursor ion scan m/z 264 in +ve ion mode is specific to identify ceramides in a sample
m/z 264
680 700 720 740 760 780 800 820 840 860 880 9000
24000 806.7
805.7
784.6804.7
783.6 807.7782.6772.6 803.7
785.6786.6
808.7771.6 781.6770.6
773.6780.6
769.6 794.6758.6 809.7828.7757.6 779.6 793.6 827.7792.6795.6 826.7 830.7759.6
829.7744.6 825.7
745.6768.6746.6 810.7 820.7760.6756.6
743.6 766.6 819.7
729.6718.6835.7734.6
Mass/Charge, Da
120 180 240 300 360 420 480 540 600 660 720 7800
2400184.0740
784.5867
785.5916
786.6022124.9972
185.0768
PC 36:3
Rel
ativ
e in
tens
ity
Precursor ion scan m/z 184.073 for PC/SM in a C. eleganslipid extract [A]; MS/MS of the precursor ion m/z 784 [B]
11
Several isobaric compounds-Identification by high resolution mass spectrometry
O
O
OH
O P
OH
OO
Nm/z 524calc. m/z 524.3711
O
O
O
O P
OH
OO
N
O
O
O P
OH
OO
N
O
O
m/z 524calc. m/z 524.3347
m/z 524calc. m/z 524.3711
Product ion spectra of deprotonated arachidonic acid [AA] and its oxidation product 5-hydroxy-eicosatetraenoic
acids [5-HETE]
30 60 90 120 150 180 210 240 270 300
8.0e4 59.04
303.28
83.08177.17 231.24 259.22163.1470.96 205.23135.06 285.28
44 Da
18 Da
20 60 100 140 180 220 260 300 340
9.0e4
Inten
sity, cps
115.059.0
203.2
167.0 257.2 261.0177.3 319.2
m/z 115.0401
COO-
Inten
sity, cps
12
220 280 300 320 360 380 m/z0
%
0
100
%
255.050
256.057
297.043
267.037
268.041281.051
307.065321.046
363.046335.061351.044 381.055
100
240 260 340
[A]
[B]
-162 Da
Yo+
-120 Da
O
OH
O
O
O
OHOH
CH2OH
OH
daidzin
Puerarin
Structure determination: Accurate mass of a precursor ion is not enough, but MS/MS is
Prasain et al., J. Agric. Food Chem., 2003
Substructure analysis in ESI-MS/MS(dereplication and partial identification
of natural products)
13
Fragmentation of basic taxoids from T.Wallichiana extract
Source: Stefanowicz 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
14
MS/MS precursor-scan spectra of typical alkaloid side chains to identify the basic taxoids compounds in an ethyl acetate
extract of T. wallichiana.
Stefanowicz et al. Anal Chem, 2001
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
Intensity
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
15
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.