Phosphorylation and Glycosylation by ECD - UAB · 2011. 3. 4. · CH 2 O HO P HO O H N 2 COOH CH 2...
Transcript of Phosphorylation and Glycosylation by ECD - UAB · 2011. 3. 4. · CH 2 O HO P HO O H N 2 COOH CH 2...
Phosphorylation and Glycosylation by ECD
Matt RenfrowSeptember 12, 2006
© 2006
H2N C C NOR1
C C NORn-1
C CORn
OH
mH+
y1
bn-1
z1·
cn-1
...
How do Peptides Cleavein the gas phase?
ECD
IRMPDCID
slow heating methods
electron radicalchemistry
H2N C C NOR1
C C NORn-1
C CORn
OH
mH+
y1
bn-1
z1·
cn-1
...
ECD
How do Peptides With LabilePTMs Cleave?
PTM PTM
IRMPDCID
H2N C C NOR1
C C NORn-1
C CORn
OH
m+nHn+
y1
bn-1
z1·
cn-1
...
ECD
IRMPDCID
NH2 CH
Facile loss of H3PO4X-P cleavage preferred Retention of
labile modificationsNo X-P cleavage
COOH
CH2
O
POH
OH
O
NH2 COOH
CH2
NH2 CH
COOH
CH2
OH-98 Da (-18 Da)
PTM
IRMPD25 scans
9-K G S E Q E S V K E F L A K-22
400m/z
700600500 800 900
[M + 3H]3+
[M - H3PO4 +3H]3+
[y13 - H3PO4 +2H]2+
[y12 - H3PO4 +2H]2+
[y11 - NH3 +2H ]2+
y102+
[M - H3PO4- H2O +3H]3+
y82+
Tryptic PKA phosphopeptides
Chalmers et al., Proteomics 2004
y
b
400m/z
1000800600 1200 1400
9-K G S E Q E S V K E F L A K-22
x 10 ~
c12p
c11p
c10p
c9p
c8p
c7p
c6p
c132+ p
C5 p
c4 pc3
p
z4•
z5•
z6•
z8•
z9•
z10•
z11•
[M + 2H]2+ &[M + 3H]2+ •
[M + 3H]3+
ECD 50 scans c
z
Chalmers et al., Proteomics 2004
RAP30RAP74
TFIIF
RNA polymerase
Dephosphorylation of RNA polymerase largest subunit
PP PPP
RNA polymerase
Initiation Elongation
multiplekinases
P
P Fcp1Phosphatase
P PP90 KDa
900 1000 1200 1300 m/z
[M + 2H]2+
[M + HPO3+ 2H]2+
1100 1400 1500700 800
Where are additions of 1st and 2nd
phosphate groups?
ESI FT-ICR MSNEDEGSSSEADEMAKALEAELNDLM
Fcp1 C-terminal fragment
Abbott, Renfrow et al., Biochemistry 2005
600 1200 m/z1000 1400 1600800 1800 2000
c6p c7
p
c5
c8p
[M+HPO3 + 3H]3+ [M+ HPO3 + 3H]2+• &[M+ HPO3 +2H]2+
N E D E G pS S S E A D E M A K A L E A E L N D L M
c9p
c10p
c11p
c222+ p
c232+ p
~ x 14
~ x 3
c13p
c14p
c18p
c19p
c20p
z5•
z7•
z11•z12
•
z13•
z14•
z18• z19
• z20• p
c17p
z17•
ECD (10 ms)
Abbott, Renfrow et al., Biochemistry 2005
13501300 1400 1450 1500
13501300 1400 1450 1500
13501300 1400 1450 1500
[M+2H]2+
[M+2H]2+
[M+HPO3+2H]2+
[M+2H]2+
[M+HPO3+2H]2+
[M+(HPO3)2+2H]2+
0 hr
2 hr
Incubation with CKIIESI FT-ICR MS
21 hr
Abbott, Renfrow et al., Biochemistry 2005
Positive ion mode ESI MS
1350 1400 m/z
ATP + kinase (4h)
[M+2H]2+
+1 pS
ATP + kinase (4h)30 min β-elimination + ethanethiol addition
1350 1400 m/z
[M+2H]2+
+ 1S*
+ 2S*
+ 3S*
Molloy, M.P., Andrews, P.C. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylatedpeptides by mass spectrometry. Anal. Chem. 2001, 73, 22, 5387-5394
Abbott, Renfrow et al., Biochemistry 2005
400 450 600 m/z550 650 700500 750 800 850
c3 c4
c5 (23/1 s/n) c7* (38/1 s/n) c8
**c6*
131 87 131
(c6) (c7**)
ν2
N E D E G S* S S* E A D E M A K A L E A E L N D L M
Abbott, Renfrow et al., Biochemistry 2005
Fcp1A phosphatase CTD phosphorylation
Abbott, K.L; Renfrow, M.B.; Chalmers, M.J.; Nguyen, B.D.; Marshall, A.G., Legault, P.; Omichinski, J.G. “Enhanced Binding of RNAP II CTDPhosphatase FCP1 to RAP74 following CK2 phosphorylation”Biochemistry, submitted
30 min
1350 1400 m/z
[M+2H]2+
+ 1S*
+ 2S*
5 h
1 h[M+2H]2+
[M+2H]2+
ControlNo kinase or ATP
Beta elimination + ethanethiol addition
time course
[M+2H]2+
+ 1S*
+ 2S*
1350 1400 m/z
4h ATP30 minβ-elim.
m/z15001450 15501400 16501600 1700 1750
GalNac
Gal
Gal
ESI FT-ICR MS of (Mce) IgA1 HRisolated from
trypsin-pepsin digestHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL
Renfrow et al., JBC, 2005
GalNAc
GalGal
1450 155015001400 1600 1650 m/z
1450 155015001400 1600 1650 m/z
1450 155015001400 1600 1650 m/z
GalNAc
Gal
Gal
GalNAcGal
GalNAc
FT-ICR-MS
quadrupole+ SWIFTisolated
IRMPDMS / MS
Renfrow et al., JBC, 2005
m/z12001100 13001000 15001400 1600 1700
m/z12001100 13001000 15001400 1600 1700neuraminidase + O-glycanase treated
ESI FT-ICR MSneuraminidase treated
GalNac
De-glycosylated IgA1 Hinge RegionGalNac
Gal
Gal
de-glycosylated HR peptide
Renfrow et al., JBC, 2005
V T V P C P V P S T P P T P S P S T P P T P S P S C C H P R L
300 500 900 m/z1100700 1300 1500
[M + 3H]3+
IRMPD
[M + 2H]2+
~ x 5
~ x 5
b3
y3
y132+/ b7
b5
y102+
y142+
y162+
y172+
y212+
y222+
y242+
y262+
y272+
y282+
y292+
y273+
y293+
Confirmed IgA1 Hinge Region sequence
- HYTNPSQD
Renfrow et al., JBC, 2005
m/z15001450 15501400 16501600 1700 1750
GalNac
Gal
Gal
GlycoMod assigned O-glycansV T V P C P V P S T P P T P S P S T P P T P S P S C C H P R L
= Gal= GalNAc4 x 4 x 5 x 4 x
5 x 5 x4 x 3 x
Renfrow et al., JBC, 2005
m/z12001000 1400800 18001600 2000 2200 2400
V T V P C P V P S T P P T P S P S T P P T P S P S C C H P R L
[M+3H]2+• & [M+2H]2+
[M + 3H]3+
~ x 40AI-ECD
= Gal= GalNAc
~ x 2~ x 2
z17
230
S
2665 2475
c16
228
T
c8c9
b11
c12
225
T
z14
1930
z15
232
S
Renfrow et al., JBC, 2005
Analyzing Phosphorylationand Glycosylation by
ECD FT-ICR MS•Some PTM’s can be so labile they are the dominant fragment in a CID MS/MS (i.e. no useful information).
•Electron Capture Dissociation (ECD) fragments peptides and proteins by a different mechanism, leaving labile PTM’s intact
•While ECD is performed in an FT-ICR MS, a new method, Electron Transfer Dissociation (ETD) is performed in a 2D and 3D ion trap (as quickly as CID in an ion trap).