lei tan, yu xia department of chemistry purdue university€¦ · lei tan, yu xia department of...
Post on 14-Jun-2020
0 Views
Preview:
TRANSCRIPT
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
top related