Lei Tan, Yu Xia

Department of Chemistry

Purdue University

1

Introduction• Sulfinyl radical (-SO•)

– Photolysis of sulfoxide (sulfur cycle)

– Observed in protein system

Gauld J.W.; J. Am. Chem. Soc. 2000, 122, 2035-2040

ArSO•CH3SO•HSO•(CH3)2SO + hv → CH3SO• + •CH3

Darmanyan A.P. et al, J. Phys. Chem. A. 1997, 101(37), 6855-6863

Inactivation of Pyruvate Formate-Lyase (PFL)

Glycyl

Cysteine

OxygenOxygen

Enzyme with Radical• Storage site: Glycyl radical • Active site: Thiyl radical

ESR of protein sulfinyl radical

2

H2O + e- •OH + H + e-

O + H2O 2 •OH

Peptide Sulfinyl RadicalPeptide Sulfinyl Radical

Helium

AC

HV: 1-2 kV

2-3 kV30 kHz

NanoESI

AP Plasma MS inlet

523.6532.1

531.6[+16]2+

x5[M+2H]2+

525 530 535 540

[M+H+Na]2+

[+17]2+C I E L L Q A R C

New Way to Form Sulfinyl Radical Ion

Xia Y.; Anal. Chem.; 2010, 82(7), 2856-2864Ma X.X.; J. Am. Soc. Mass Spectr. 2011, 22(5), 922-930

CCSH

CO2HH2N SO•

n•++nH

n++nH

CO2H

SO•Oyn

HbmC

HS

H2N C+nH+

n•+

CID

radical rxns(e.g. •OH)

3

Formation of Site-Specific Sulfinyl Radical

ESI

radical rxns(e.g. OH•)

Inter-chain disulfide linked peptide Site-Specific

Peptide Sulfinyl Radical Ion

COOHCOOHH2NH2N

COOHCOOHH2NH2N

COOHH2N

COOHH2N

+/-COOH

H2N

+COOH

H2N

+/-

• Easy to determine the site of sulfinyl radical• Change the polarity of ions easily.• Easy to determine the site of sulfinyl radical• Change the polarity of ions easily.

Chain A

Chain B

Chain A/B

4

Experiment Setup

Wed. PosterWP 760

4000 QTRAP

Ion/RadicalReaction

Q0 Q1 Q2 Q3

2. Low Pressure Hg Lamp –185 nm , 248 nm

20 mA

NanoESIAI foil

MS inlet

1. Helium Atmospheric Pressure (AP) Low Temperature Plasma

Craig Stinson5

Formation of Site-Specific Peptide Sulfinyl Radical Ions

6

[ CLPTR+H ]•+_

SO•[ASO•+H]•+

[ASH+H]+ [ CLPTR+H ]•+_SH

Sulfinyl Radical

0

50

100

[M+2H]2+

524.5

589.5[ASH+H]+

476.3[BSO•+H]•+461.3

[BSH+H]+

450 550 650600500

621.4 637.4

[M+Na+H]2+

[M+K+H]2+

Formation of Site-Specific Peptide Sulfinyl IonCLPTR

HMAC

_

Chain B: 459.2

Chain A: 587.3 Positive mode

604.4[ASO•+H]+

7

0

50

100

[M+2H]2+

524.5

589.5[ASH+H]+

476.3[BSO•+H]•+461.3

[BSH+H]+

450 550 650600500

621.4 637.4

[M+Na+H]2+

[M+K+H]2+

Formation of Site-Specific Peptide Sulfinyl IonCLPTR

HMAC

_

Chain B: 459.2

Chain A: 587.3 Positive mode

m/z

Negative mode522.4

474.4[BSO•-H]•-

459.3[BSH-H]-

[M-2H]2-

491.4602.5

[ASO•-H]•-587.5

[ASH-H]-

619.40

50

100

450 550 650600500

604.4[ASO•+H]+

8

580 590 600 610 620 630Incre

asing Flow Rate

m/z

Formation of Sulfinyl Radical Ion

Higher Helium Flow Rate More Intense PlasmaHigher Radical Density

589.3

604.3

621.3

Consecutiveoxidation[ CLPTR+H ]+_

SO•

[ CLPTR+H ]+_

SH

a)

b)c)

9

Cys-SO•, 137 m/z

Protonation EnergyNH2 0 kJ/molC=O +112.9 kJ/mol

*All calculation was carried out using Gaussian, MP2/6-31G* andB3LYP/6-31G*

Cys-SO• Spin densities

Neutral S: 0.510O: 0.488

Protonated at NH2S: 0.543O: 0.456

Protonated at C=O S: 0.552O: 0.453

Structure of Cysteine Sulfinyl Radical Ions

Joe FranciscoChasity LoveTue. Poster: TP 761

10

Collisional Induced Dissociation (CID) of Peptide Sulfinyl Radical Ions

11

- Radical v.s. Charge Driven Dissociation

- 62Da (CH2SO)

- 62Da (CH2SO)- 62Da (CH2SO)

Ion trap CID of Peptide Sulfinyl Cation

400 500 600 200 300 4000

50

100317.2

379.2

330.20

50

100542.3

604.3

586.3486.2

250 350 4500

50

100414.2

476.2

427.1

-49Da - HSO

-18Da

-56Da(L)

-18Da

-HSO

-74Da(M)

-47Da(M)

b2+

[ CLPTR+H ]+_

SO•

[ GCGK+H ]+_

SO•

[ HMAC+H ]+_

SO•

Rel

. Int

. (%

)

m/z

(a) (b)

(c)

12

2

HN

CHC

O2

CID +62 Da

Glycyl Radical Accurate Mass

61.9826 DaAccurate Mass

61.9826 Da

Loss of 62 Da (CH2SO)

Activation energy: 38.7 ± 1.4 kcal/mol* Calculation based on neutral cysteine sulfinyl radical

Activation energy for amide bond cleavage: 25-40 kcal/mol

Sulfinyl Radical

Lioe H.; J. Am. Soc. Mass Spectr. 2007, 18, 1109-112313

Radical Directed DissociationRadical Directed Dissociation

-62Da (CH2SO)

- 62Da (CH2SO)

- 62Da (CH2SO)- 62Da (CH2SO)

Ion trap CID of Peptide Sulfinyl Cation

400 500 600 200 300 4000

50

100317.2

379.2

330.20

50

100542.3

604.3

586.3486.2

250 350 4500

50

100414.2

476.2

427.1

0

50

100

351.2

-49Da - HSO

-18Da

-56Da(L)

-18Da

-HSO

-74Da(M)

-47Da(M)

b2+

a2+

b2+

[ CLPTR+H ]+_

SO•

[ GCGK+H ]+_

SO•

[ HMAC+H ]+_

SO•[ TLC+H ]+_

SO•

Rel

. Int

. (%

)

m/z m/z

(a) (b)

(c) (d)

100 200 300 400

215.1

187.1

143.0

14

- 62Da (CH2SO)

Radical v.s. Charge Directed Dissociation- Charge States

100 300 500 7000

50

100

647.4

585.4

-56Da(I or L)

100 300 500 7000

50

100324.2

y4+

y3+

y2+

ob3+

y1+

ob4+-62Da

m/z

[ C G A I L R+ 2H ]2•+_SO•

[ C G A I L R+ H ]•+

SO•

_

[M+2H]2+

[M+H]+

Radical DirectedDissociation

Charge DirectedDissociation

• Loss of CH2SO~ 39 kcal/mol

• Amide bond cleavage25-40 kcal/mol

15

- CH2SO

- 49Da (HSO)

N-terminal Acetylation

592.3

[ CH3-CO-C G N K R+ H ]•+_

SO•

400 500 600

400 500 600

585.4

634.3

526.3

572.4

530.3- 62Da (CH2SO)

[ C G N K R+ H ]•+_

SO•

Acetylation can promote 49Da (HSO) loss!Acetylation can promote 49Da (HSO) loss!m/z

+ 42 Da

16

100 200 300 400

100 200 300 400

m/z

Charge/Radical Location

100 200 300 400

317.2 379.2

330.2246.1

199.0 228.1 361.1299.1129.0

[ GGCK + H ]•+_

SO•

317.2

379.2

330.2246.1 361.1

- 49Da (HSO)-71Da(K)

287.1

- HSO

- CH2SO

[ GCGK + H ]•+_

SO•

[ CGGK + H ]•+_

SO•

-71Da(K)

317.2

379.2

- CH2SO

- 62Da (CH2SO)

17

100 200 300 400

Loss of 49Da (HSO•)

199.0

330.2

295.1

312.2216.1y2

+

*y2+

[ G G C K - HSO + H ]+_

SO•

m/z

But somehow affected by charge…

dehydroalanine

18

Charge Polarity

100 200 300 400

318.2

380.2

283.1175.1

329.2

378.2

361.2312.2

100 200 300 400m/z

[ CSR + H ]•+_

SO•

[ CSR - H ]•-_

SO•

- 49Da (HSO)

- 62Da (CH2SO)

19

Reactivity of Sulfinyl Radical

- The fate of sulfinyl radical- Ion/molecular reaction

20

Experiment Setup

4000 QTRAP

Ion/RadicalReaction

0.05 mTorr5.0 m Torr

Q0 Q1

Q2

Q3

Ion-moleculereaction

NeutralCAD gas line

21

[ CLPTR + H ]+_

SO•

600 650 700 750m/z550 800

604.2

6956050.0

3.5 645.1+ CH2-CH=CH2 (41 Da)

595.1573.0

Trapping 3s with ally iodideTrapping 3s with ally iodide

Ion-Molecule Rxns of Peptide Sulfinyl Radical Ions

+ I+

+ CH2-CH=CH2

22

600 650 700 750m/z550 800

604.2

595.1

Trapping 3s with dimethyl disulfideTrapping 3s with dimethyl disulfide

619.4621.4 651.4

+ SCH3 (47 Da)+ 15 Da

6956050.0

1.5

Ion-Molecule Rxns of Peptide Sulfinyl Radical Ions

[ CLPTR + H ]+_

SO•

+ +

23

[ CLPTR + H ]+_

SO•

Ion-Molecule Rxns: Sulfinyl v.s. Thiyl

600 650 700 750m/z550 8000%

5%

10%

[ CLPTR + H ]+_

S•

600 650 700 750m/z550 800

604.3

588.3

635.3+ 47 Da (SCH3)

711.3+ 123 Da (SPh)

Trapping 3 s with CH3-S-S-Ph

0%

5%

10%

651.3+ 47 Da (SCH3)

24

2

HN

CHC

O2

CID +62 Da

Glycyl Radical

Formation of Site-Specific Glycyl Radical

Sulfinyl Radical

25

[GGGK + H]+•

[GGGK + H]+•

[GGGK + H]+•

100 200 300 400

-71(K)

y3+1 -18

100 200 300 400

261.1317.2

204.2186.1

299.2129.1

y2

-71(K)

204.1y2+1

y3

m/z

Sulfinyl Radical

Glycyl RadicalCID

CID

261.1

317.1

287.1246.1 299.1204.1186.1129.1

y3+1

y2

-30-71(K)

100 200 300 400

317.1

246.1

299.1261.1228.1

Tracking Radical Migration

CH2=CH(CH2)2NH271 Da

26

Summary

CID

• Basic amino acid• Charge state• Charge location• Charge polarity

HN

CHC

O

Glycyl Radical

- CH2SO

Ion-moleculereaction

• ‐S‐S‐• CH2=CH‐CH2‐ILow reactivity 

Sulfinyl Radical

ESI

radical rxns(e.g. OH•)

COOHCOOHH2NH2N

COOHCOOHH2NH2N

COOHH2N

COOHH2N

COOHH2N

+/-

27

Acknowledgement

Dr. Larry CampbellDr. Jim HagerDr. Joe Francisco

28