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.