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).