Post-translational modifications

PC235Katalin F. Medzihradszky

folkl@cgl.ucsf.edu

Post-translational Modification of ProteinsExpanding Nature’s Inventory (2006)by C.T. Walsh

ISBN 0-9747077-3-2

Genome↓

Transcription – mRNA↓

Translation – proteins + co-translational modifications

↓Post-translational modifications

Just for introduction

Post-translational modifications I• Enzymatic processing• N-, O-, C-linked glycosylation – Asn, Ser/Thr/Hyl/Hyp,

Trp• Phosphorylation – Tyr, Ser, Thr, His, Asp• Acylation

acetylation of the N-terminusfatty acid anchors on Cys

• Cross-linkage – Lys, Trp, Tyr, Met• Oxydation – Cys, Met, Trp, Tyr, His• Methylation – N-terminus, Arg, Lys• Ubiquitination - Lys

Post-translational modifications II

• Cannot be predicted – though consensus sequences have been reported for some of them

• Organism-dependent• Can be tissue- or location-specific• Stable or dynamic – high and low level• Alters biological activity, and physical

properties• May alter the immune response

↓

Sample preparation

• Will the modification survive?

• Can I get it down to a mass spectrometry friendly size?

• Isolation/enrichment?

• Losses – too hydrophilic/hydrophobic

Mass spectrometry

• Will the modification survive the ionization? the MS/MS activation?

• How unambiguous is the assignment? modification assignment?

Ac vs. Me3; phosphate vs. sulfate etc. site assignment?

Finding the N-termini

• 80-90% of the eukaryotic proteins is acetylated

If 2nd aa: G, A, S, C, T, P, V - Met-1 is clipped off; Ac- is added to G,

A, S, TIf 2nd aa: E, D, Q, M, I, L, W, F –persisting Met gets acetylated

Finding the N-termini

• McDonald L, et al., Positional proteomics: selective recovery and analysis of N-terminal proteolyticpeptides.Nat Methods. 2005 Dec;2(12):955-7. Epub 2005 Nov 18.

Modified N-terminus

Modified side-chain

I don’t think any search engine will tell the difference !

Methylation (mono, di, tri - +14, +28, +42 Da)

• N-terminus, Lys, Arg, His• Trimethyl – acetyl = 36 mmuAccurate mass measurement helps;Fragmentation is different too

• Glu (Asp) may form Me-ester – upon CBB staining (MeOH + acid) + 14 Da

7000

6000

5000

4000

3000

2000

1000

0

644.0643.8643.6643.4643.2643.0642.8642.6m/z

14

12

10

8

6

4

2

0

x103

795.0794.8794.6794.4794.2794.0793.8793.6m/z

642.77672 (10+)

b57 + 42 Da

Calculated masses 6418.6718 - with Ac 6418.7082 - with Me3

Experimental value 6418.6973 Me3b57 (-1.6 ppm)

Mass calculated: 7134.8569 Experimental: 7134.8463 (-1.4 ppm)

y64

793.65655 (9+)

FT ICR MS

700

600

500

400

300

200

100

0

Ion

Cou

nts

400350300250200150100

m/z100

80

60

40

20

0

Ion

Cou

nts

800700600500400

m/z

72.08

87.05147.11 169.10

186.13

214.13

260.20

280.20(2+)

294.85(3+)308.86(3+)

329.74(2+)

373.26(2+)

745.50

623.36599.39

462.79(2+)

479.83(2+)

571.28

486.31

422.77(2+)

427.77(2+)

441.

54(2

+)

458.

29

471.29(2+)

314.5(3+)

320.2(3+)

354.73(2+)

141.1

V

N y1

SV-28-H2O

a2-NH3

a2

b2

y2

y42+

[MH3-NH3-NMe3]3+

[MH3-2NH3]3+

[MH3-NH3]3+

y52+

[a7-NH3-NMe3]2+ y6

2+

y72+

[MH2-CO2H-NMe3]2+

[MH2-NH3]2+

MH22+

SVK*ESVK*EI

SVK

*E-2

8

[MH2-2xNH3]2+

y6b6-NH3-NMe3

512.36

SVK*EI-28

SVK*EI-28-NMe3

Asn-Val-Ser-Val-Lys(Me3)-Glu-Ile-Lys

- 59 Da

Disulfide-bridges

• in membrane and secreted proteinsimportant 3D structure feature

• prone to shuffling @ basic pH

Digestion

@ low pH

Reduction/alkylation

Reduction/alkylation

I

m/zI

m/z

I

m/z

I

m/z

I

m/z

MS/MS

Reduction/alkylation

Digestion

@ low pH

Reduction/alkylation

Assigning disulfide-bridges

Synthetic Ac-TIMP-1(Ser175)126-184

ECTVFPCLSI PCKLQSGTHC LWTDQLLQGSEKGFQSRHLA CLPREPGLCS WQSLRSQIA

Where are the disulfide bridges?

* digestion with trypsin @ pH 6;

* with pepsin in acid

Bodi, N. et al., J. Pept. Sci. 9, 430-441 (2003).

300028002600240022002000m/z

inte

nsity

12001000800600m/z

inte

nsity

1600140012001000800600m/z

[38-

44]

free

SH

[33-

37]

[45-

55]

Free

SH

[38-

55]

41-4

9

[38-

44]-[

45-5

5]41

-49

Ac[

1-13

]-[45

-55]

2-7,

12-

49

[14-

32]-[

38-4

4]20

-41

[33-

37]

[38-

44]

S*

[45-

55]

S*

Ac[

1-13

]1

S-S,

1 S

*

Ac[

1-13

]3

S*[4

5-59

]1

S*

30002800260024002200200018001600m/z

Ac[

1-13

]1f

ree

SH

, 2 S

*

[38-

55]

2 S*

[14-

32]

1 S*

[38-

59]

2 S*

[14-

37]

1 S*

MALDI-TOF analysis of the tryptic digest

PSD

In-source reduction

Results from the MALDI-TOF MS

594.30 [33-37] 809.5 [38-44] szabad SH1275.7 [45-55] szabad SH2064.0 [38-55] 41-49 2082.1 [38-44]-[45-55] 41-49 2752.4 Ac[1-13]-[45-55] 2-49, 7-12 2950.6 [14-32]-[38-44] 20-41 3602.6 Ac[1-32] 2-7, 12-20 3620.6 Ac[1-13]-[14-32] 2-7, 12-20 4177.3 Ac[1-37] 2-7, 12-20 5665.1 Ac[1-32]-[37-

55]+H2O 2-49, 7-12, 20-41

5683.2 Ac[1-32]-[37-55]+2 H2O

2-49, 7-12, 20-41

594.30 [33-37] 866.5 [38-44] 1 CM Cys 1332.7 [45-55] 1 CM Cys 1536.8 Ac[1-13] 1 S-S, 1 CM Cys 1595.8 Ac[1-13] 1 SH, 2 CM Cys 1652.7 Ac[1-13] 3 CM Cys 1674.8 [45-59] SH 1731.9 [45-59] 1 CM Cys 2144.2 [14-32] SH 2180.1 [38-55] 2 CM Cys 2201.1 [14-32] 1 CM Cys 2579.3 [38-59] 2 CM Cys 2776.3 [14-37] 1 CM Cys 3834.7 Ac[1-32] 4 CM Cys 4409.7 Ac[1-37] 4 CM Cys

Tryptic digest After reduction/alkylation

Disulfide bridges in TIMP-1 C-terminal domain: [38-44]-S-S-[45-55], PSD of MH+ at m/z 2082.1

1000

800

600

400

200

0

Inte

nsity

200015001000500m/z

2X17

5.6,

y1,

y 1*

251.

6, b

2

255.

2, y

2*-N

H3

322.

6, b

3

776.

3 810

.084

1.7 1

242.

1*12

76.4*

1308

.9*

110.

1, H

1928

, b6+

H2O

4, b

10*+

H2O

His-Leu-Ala-NH-CH-CO-Leu-Pro-Arg

CH2

CH2

Glu-Pro-Gly-Leu-NH-CH-CO-Ser-Trp-Gln-Ser-Leu-Arg*

S

S810

776

842.11242.4

1276.51308.5

38

45

MALDI-PSD/CID yields characteristic triplets

4000

3000

2000

1000

0

Ion

Cou

nts

600500400300200100

m/z1200

800

400

0

Ion

Cou

nts

1000900800700600

m/z

400

300

200

100

0

Ion

Cou

nts

1600150014001300120011001000

m/z

Figure 9. Low energy CID of m/z 871.79 (most abundant ion in 9+ cluster), MW (monoisotopic) 7835.03 Da. This molecule was identifiedas disulfide linked peptides [443-496] and [519-531] of myosin heavy chain. Sequence and fragmentation are shown on the next page.

327.21y3**

456.25y4**

490.24y4 569.34

y5**

120.08F

201.12b2

637.32y5 682.44

y6**763.10 (3+)

b19

811.49y7**

796.13(3+)b20

872.18 (3+)b22

706.41 (3+)b17

924.5y8**

1144.16 (2+)

1307.77 (2+)

1068.62b9 b19

b22

No characteristic triplets in ESI-CID

Fragment ions used for the identification of disulfide linked myosin peptides. (see low energy CID spectrum in Figure 9.)

b-ions myosin heavy chain peptide [443-496] 763.10 (3+) 1144.16 (2+) 796.13 (3+) 872.18 (3+) 201.12 1068.62 706.41 (3+) 1307.77 (2+) 2 9 17 19 20 22 VTRINQQLDTKQPRQYFGVLDIAGFEIFDFNSLEQLCINFTNEKLQQFFNHHhS 5 4 637.32 490.24 y-ions myosin heavy chain peptide [519-531] DLAACIELIEKPhS 8 7 6 5 4 3 924.5 811.49 327.21 682.44 456.25 569.34 **y-ions

Disulfide BridgesThe ProteinProspector mass modification search can be used in conjunction with MS-Bridge to find peptides with disulfide bridges. For this example, mass shifts between 0-2000 Da were considered. (1023.3276+4).

KLSWADLIVFAGNCALESMGFK+4

VSFADLVVLGGCAAIEK

Glycosylationhttp://glycores.ncifcrf.gov/

Reference: Essentials of Glycobiology by Varki et al.

N-linked

AsnXxxSer/Thr/Cys

Further processing

N-linked glycosylation• consensus sequence• GlcNAc2Man3 – coreoligomannose structure – just Man unitscomplex sugars– GlcNAc-Gal–SA antennaehybrid structurescore fucosylationsulfate, phosphate modifications

• PNGase F removes all N-linked structures; Asn Asp

N-linked glycosylation• Incredible heterogeneity: a site may be

only partially occupied and may display numerous different carbohydrates

• species-, tissue-, cell-type-specificmodification, physiological changes, diseases may alter the sugars

certain structures are immunogenicGal 1-3 capping, Fuc 1-3 on inner GlcNAc;blood group determinants

N-linked glycosylation• Identification from diagnostic fragments:* HexNAc m/z 204* HexHexNAc m/z 366 Precursor scan, or „ping-pong” acquisition• Identification from oligosaccharide

heterogeneity• enrichment by HILIC or lectin-

chromatography

human lecithin:cholesterol acyltranferase and apolipoprotein D, tryptic digest, LC/MS analysis

MHTVNGYVN*R

Recombinant Factor VIII, 50 kDa subunit

AG(Man8GlcNAc2)NVSNIIPASATLNADVR peptide+GlcNAc

About the structures of N-linked glycopeptides

• from the measured mass, and the CID spectrum the modified peptide can be identified + the size and class of the sugar

• the identity of the sugar units and their linkage positions CANNOT be determined

• NMR, exo- and endoglycosidases are needed to complete the job

[MHNa2]3+ of QV(Man10GlcNAc2)NIT and [MH2Na] 3+ of QV(Man9GlcNAc2)NITGK

Scheme 1. Fragments observed in the low energy CID spectrum of [MH2Na]3+ of glycopeptide QV(Man9GlcNAc2)NITGK (Figure 6)

+Na 2159.81 1080.41(2+) +Na 671.18 +Na Gln Man1-2Man1 1997.77 999.39(2+)

36

Man1 Val Man1

Man1 36

Man1-4GlcNAc-GlcNAc-Asn 6 Man1-2Man1- 2Man1 Ile +Na +Na Thr 163.05 2483.87 1242.44(2+) 1684.46 2321.851161.43(2+) 962.49 Gly +Na 347.08 Lys +Na 1360.41 +Na Man1-2Man1 1036.30

36

Man1 Man1

Man1 36

Man1-4GlcNAc 6 Man1-2Man1 - 2Man1 204.08 +Na 1522.41 +Na 1198.36

One component from the previous slide

K.F. Medzihradszky Meth. Enzymol. 405, 116-138 (2005).

O-linked sugars• No consensus sequence• No common core structure• No universal enzyme-elimination works (NaOH)sugars have to be reduced upon release

Detection is problematic – because of heterogeneity; variable site occupancy

Site assignment is even harder

O-linked sugars

Other O-linked core structures

• FucHarris, R.J. & Spelmann, M.W. (1993) Glycobiology, 3, 219-224.• GlcNishimura, H et al., (1989) J. Biol. Chem. 264, 20320-20325.• Man – in yeast____________________________________

• GlcNAc – single unit; INSIDE the cell

CID fragmentation

of O-linked glycopeptides

J. Am. Soc. Mass Spectrom. 7, 1996, 319-328.

O-linked GlcNAc

•Regulatory modification of nuclear and cytoplasmic proteins•Poorly understood due to lack of effective methods for enrichment and detection.

The Enrichment Problem•WGA lectin has affinity for GlcNAc, but affinity to a single GlcNAc moiety is low: millimolar1.

WGA

Good recoveryof glycoprotein

Complex glycosylation

Ohlson S et al. Bioseparation (1998) 7 p.101

GlcNAcGlcNAcGlcNAc

protein

No recovery

WGAPSVPVSerGSAPGR

O-GlcNAcO-GlcNAcylation

Load Wash Elute

Load Wash Elute

O-GlcNAc Enrichment

WGA column fraction collection

Isocratic ChromatographyPeptide Mixture

Non-enriched

O-GlcNAcmodifiedenriched

UV

abs

orba

nce

214

Time (minutes isocratic HPLC)

Vosseller, K. et al. Mol Cell Proteomics (2006) 5 5: p.923-934

•Selective Enrichment of O-GlcNAc modified peptides using lectin weak affinity chromatography1.

CID Analysis of O-GlcNAc-Modified Peptides

Chalkley, R. J. and Burlingame, A. L. J. Am. Soc. Mass Spectrom. (2001) 12 p.1106-1113

•O-glycosidic link is significantly more labile under CID conditions than peptide backbone.

•Modification site identification using CID often not possible.

A bit about MS/MS alternatives

• ECD (electron-capture dissociation) –multiply charged ions meet electron beam in FT-ICR – larger the charge state larger the capture’s efficiency

• ETD (electron-transfer dissociation) –multiply charged ions meet stable anion (fluoranthene) in ion traps

• radical ion is formed, different mechanism mostly backbone cleavages

Mechanism of ECD I

NN

H

R1

O

H O

N

H

NN

H

R1

O

H O

NH

+ 2H

3+

+ 2H

2+

NN

H

R1

O

H

CH2CH2

CH2NH2

O

NH

+ 2H

2+

NN

H

R1

O

H

H

+ H

+O

N

HCH2CH2

H2CNH2

+ H

+

c` ion z ion

+

e

electroncapture

backbone cleavage/proton abstraction

H2N H2N

H

H H

Mechanism of ECD II

NN

NN

OH

H O

Ri + 1 H

Ri

H

O

Ri - 1

H

+ 2H

2+

NN

N

OH

HRi

H

O

Ri - 1

H

N

O

Ri + 1 H

+ H

+ H

+

+

c` ion

z ion

NN

H

O

Ri - 1

H

NN

OH

H O

Ri + 1 H

Ri

+ H

+ H

+

+

c ion

z` ion

Bakken, V.; Helgaker, T.; Uggerud, E., Eur. J. Mass Spectrom. 2004, 10, 625-638

ECD MS Spectrum of GlcNAc-modifiedPeptide from Spectrin

Vosseller, K et al. Mol Cell Proteomics (2006) 5 5: p.923-924

One of the most important regulatory events:

turns proteins on and off

induces or prevents other post-translational modifications

in the same protein

signaling pathways : phosphorylation cascades

PhosphorylationBiological significance

Difficulties

a) Dynamic process : kinase vs. phosphatase

– both must be blocked during isolationb) Phosphorylation often @ low level (

A whole cell lysate with 20,000 sites of phosphorylation

at 1% stoichiometry

A whole cell lysate with 20,000 sites of phosphorylation

at 1% stoichiometry

phosphopeptide relative distribution after a 10,000 fold enrichment

Confirming the presence of phosphorylation

Western blot

pTyr large enough for sequence independent

recognition - works well

pSer, pThr – not reliable

Dyes – questionable reliability

Phosphatase treatment + isoelectric focusing – pI shift

In vitro/in vivo assay with radioactive phosphate

Determining site of phosphorylation

Mikrosequencing

Mutation studies

Mass spectrometry

MS spectrum : + 80 Da shift

MS/MS fragmentation :

pSer, pThr - H3PO4 loss : –98 Da

pTyr – always retains the modification

immonium ion at m/z 216 Da

Enrichment methods

Ion exchange on SCX

IMAC : Fe(3+), Ga(3+)…

binding at low pH

methyl-esterification prior to IMAC

TiO2, ZrO2

Immunoprecipitation (only for pTyr)

Large scale vs. 1 protein

large scale phosphorylation studies: large amounts of a complex mixture is analyzed

compensating for losses during sample preparation leads to more PTM identification but low end results are incidental

single protein samples: sample amount is usually limitedmore challengingcombination of techniques may be necessary

Survey Scan:

Precursor Ions that produce 79(-)

Phosphorylation Discovery Workflow

Acquire MSMS Spectra (+)

+ / -polarity switch

+ / -polarity switch

Dynamic Exclusion

N2 CAD Gas

linear ion trap

Detector

Q0 Q1 Q2 Q3/LITQ0 Q1 Q2 Q3/LIT

C2B C2B

4000 Q TRAP

Quadrupole Collision Cell

Sample

LCMS 300fmole Fetuin: Discovery and Identification of CDSSPDpSAEDVR

79- TIC

79- Spectrum at 11.1 min

Enhanced Resolution +ve ion Scan

+ve ion MSMS Spectrum

CDSSPDpSAEDVR

MH2+

MH2+-H3PO4

MALDI-TOF MS without enrichment

1200 1700 2200 m /z

0

5000

10000

15000

20000

25000

a.i.

/O =/bruker/2006/06novem ber/ke061110_02/2S R ef/pdata/1 A dm inistrator F ri N ov 10 11:56:05 2006

P P

PP

Enrichment with TiO2

1400 1900 2400 m /z

0

10000

20000

30000

40000

50000

60000

70000

a.i.

/O =/bruker/2006/06novem ber/ke061109_01/2S R ef/pdata/1 A dm inistrator F ri N ov 10 10:15:26 2006

P

P

P

P

P P

P

P

P

CID of CDSSPDpSAEDVR140x103

120

100

80

60

40

20

0

Inte

nsity

800600400200m/z

40x103

30

20

10

0

Inte

nsity

140012001000800600400200m/z

456.

7 M

-983

+

518.

3 y

4

389.

3 b

3

324.

1 b

2

274.

3 y

2

213.

1 P

D

175.

1 y

1 435

.9 y

8-98

2+

597.

2 P

DpS

AED

-98

695.

2 P

DpS

AED

498.

2 b

4

871.

4 y

7

477.

3 M

## 3

+

1094

.4 b

10-9

8

870.

5 y

7

968.

4 y

8

1192

.4 b

10

389.

2 y

3

324.

1 b

2

274.

2 y

2

462.

2 b

42#

715.

4 M

## 2

+

1360

.6 M

-py

684.

4 M

-982

+

1262

.5 M

-py-

98

756.

3 y

6

658.

4 y

6-98

m/z 733.7 (2+)

m/z 489.6 (3+)

+80 Da: phosphate? sulfate?Phosphorylation• Tyr, Ser, Thr, (His)• Phosphopeptides are stable in MS (except His)• Tyr – no phosphate loss in CID• Ser, Thr – H3PO4 (98 Da) loss in CID

Sulfation• Tyr only• Significant SO3 (80 Da) loss even in MS

FTMS accurate mass measurementwill tell the difference

Ion detected MH+ determined sequence MH+ calculated [ppm] 501.27454(3+) 1501.808 GKFDYNTFVGI/LI/LK 1501.8055 +1.6 512.26184(4+) 2046.0239 AcAEEKQGRHTTNVI/LSMFR 2046.0191 +2.3 516.26027(4+) 2062.0176 AcAEEKQGRHTTNVI/LSM(O)FR 2062.0140 +1.7 603.31016(2+) 1205.6125 HTTNVI/LSMFR 1205.6101 +1.9 611.30791(2+) 1221.608 HTTNVI/LSM(O)FR 1221.6050 +2.4 643.28905(2+) 1285.5703 sulfo-HTTNVI/LSMFR 1285.5669 +2.6 phospho-HTTNVI/LSMFR 1285.5764 -4.7

myosin regulatory light chain, Lymnaea stagnalis

Chromatographic and MS-behavior20x103

15

10

5

0

Ion

Cou

nts

30282624222018

5000

4000

3000

2000

1000

0

Ion

Cou

nts

30282624222018Time [min]

250

200

150

100

50

0

Ion

Cou

nts

580560540m/z

400

300

200

100

0

Ion

Cou

nts

580560540m/z

m/z 546.75-546.82

m/z 586.75-586.82 546.79(2+) 586.77(2+)

546.79(2+)

586.77(2+)

YASQLNQLRunmodified

+80 Da +80 Da

CID comparison of phospho- and sulfo-LAGLQDEIGSLR

100

80

60

40

20

Ion

Cou

nts

12001000800600400200

2000

1500

1000

500

0

Ion

Cou

nts

12001000800600400200m/z

627.39(2+)

676.41(2+)

185.13

242.17

288.21

327.25

355.24

414.24b2

b3

y2

a4

b4*y4

512.26

y4

771.43869.40

980.38

899.53

1069.541167.55

*y10 y10

*y8

y8-NH3y7*y7

[MH2]2+

precursor ion

[MH2-H3PO4]2+

185.13

242.15

327.25

432.26

545.35

636.39(2+)

674.42

789.44917.50

1087.61

1158.66

precursor ion - m/z 676.3(2+)

b2

b3

a4

**y4

**y5**y8

**y7

**y6**y10

**y11

[MH2-SO3]2+

1030.60

**y9

fragment mass = unmodified +80 Dafragment mass = unmodified -18 Da

fragment mass = unmodified

PHOSPHO

SULFO

Sulfopeptides easily can be identified as phosphopeptides!

• + 80 Da, 9 mmu difference only

• “identical” behavior by ESIMS, chromatography and under basic conditions.

• different CID fragmentation K.F. Medzihradszky et al., Mol. Cell. Proteomics, May 2004; 3: 429 - 440.

An exciting new cofactor

Reza GhiladiPaul Ortiz de Montellano

Catalase-peroxidase

Removes harmful peroxide molecules2 H2O2→ H2O + O2

CatalaseH2O2 alternately oxidizes/reduces the heme Fe

Peroxidaseheme is oxidized by H2O2;reduction involves H-donor, such as NADH

Crystal structure of catalase-peroxidaseHaloarcula marismortui (Yamada et al. Nature structural biology)

Active site structure

Active site structure We studied the Mycobacterium tuberculosis

enzyme1) Ghiladi RA,et al., J Biol Chem. 280, 22651-63 (2005). 2) Ghiladi RA, et al, Biochemistry, 44, 15093-105 (2005). 3) Ghiladi RA, et al., J Am Chem Soc. 127, 13428-42 (2005).

CH2 OH

- CH2 - S+ – CH3

Met-255

Tyr-229

NH

CH2 -

Trp-107

What are we looking for?

* Cross-linked “tri”-peptide calculated neutral (zwitter ionic) mass: 6880.31 Da

“measured”mass: 6879.99 Da

MS TIC

983.87(7+)

9+ 8+7+

6+

ox. (+16)

CID spectrum of the “tri”-peptideprecursor: 984.4(7+); intensities magnified by ~7.5

80

60

40

20

0

Ion

Cou

nts

140012001000800600400200

m/z

86.10

201.12229.12

288.20

358.17

472.24(2+)

493.71(2+)

550.25(2+)

671.99(3+)

449.18(2+)

703.33802.40

943.48

986.02(2+)

1217.77(4+)

1466.67(3+)

600

500

400

300

200

100

0

Ion

Cou

nts

674.0673.0672.0671.0

m/z

671.99(3+)

Fragmentation scheme of the cross-linked structure

*4397 *4116 1537* 846 644* 502 288 *4511 *4187 1820* 1594* 1155* 943* 715 573 403 175 D L E N P L A A VQMGLI-NH-CH-CO-V N P E G P N G N P D P M A A A V D I R 358 569 753 923 3712 3925 229 472 682 824 (496) 175 (567) 338 M A-NH-CH-CO-H A A G T Y R CH2 CH2 4868.31 (+H) CH3 2014.07 (-H) S + OH CH2 CH2 1728* 915* 703 589 351 147 1028* 802 * 646 452 204 NH2-CH-CO-A M N D V E T A A L I V G G H T F G K 6 1 4 91 5 * 12 12 * - 1 8 1 0* 7 4 3 98 6 * 1 3 11 * 1 56 2* 8 4 4 * 10 9 9* 1 3 6 8 * 16 6 3 *

*4511 – y-type ions, cleavage from 4868 – mostly triply charged ions detected 3712 – b-type ions, cleavage from 4868 614 – b-type ions, derived from 915* - doubly charged ion was detected 915* – singly charged ion was detected too

NH

Proven by MS/MS/MS

When we don’t have a clue…

Prospector will search for non-specified modifications

2 step search:a)what’s there?b)is it modified?

Characterization of protein populations

• structural information obtained from peptides

→ conclusions about the proteins

• Analysis of the intact proteins yielding extensive structural information

“top-down” approach

Requirements for Top-Down Analysis

• Reasonable amount of protein – pmoles.• Protein compatible with ESIMS.• Relatively simple protein mixture.• High resolution and mass accuracy instrument - FTMS• Efficient protein fragmentation: ECD > CID

• Good ion statistics and deconvolution software (needs to be able to predict isotope pattern to determine monoisotopic mass).

Histones – Ideal samples?• Small proteins

• Post-translationally extensively modified: phosphorylation, acetylation, methylation, ubiquitination

• Histone code: PTM state regulates gene transcription ‘on’and ‘off’ state

Tetrahymena histone H2B variants studied

Strahl and Allis, Nature (2000) 403, 41-45.

`Histone Code' Hypothesis

Tetrahymena histone H2B 12+ envelope

H2B.1

H2B.1+Me

H2B.1+2Me

H2B.1+42

H2B.1+42+O

H2B.1+70

H2B.1+84H2B.2+42

H2B.2+56

H2B.2+70H2B.1+3x42H2B.2+2x42

Electron-capture dissociation spectrum of the H2B mixture

Same ECD spectrum – “zoom-in”

Massobserved Assignment (Ac/Me3; 2Ac/AcMe3/2 Me3) Δ[ppm]with Mass Calculated

356.2667 c3+ 42 356.2298 /356.2661 +104/ +1.6526.3735 c4+ 2x42 526.3354/526.3718/526.4082 +72/ +3.2/ -66 723.4889 c7+ 42 723.4518/723.4882 +51/ + 0.9754.3535 z7 54.3497 -5 • • • • •

1065.6380 c11+ 42 1065.6066/1065.642 +29.5/ -3.71107.6556 c11+ 2x42 1107.6171/1107.6525/1107.6889 +34.8/ +2.7/ -30.11167.6792 *c11+ 2x42 1167.6372/1167.6736/1167.71 +36/ +4.7/ -26.41193.7422 c12+ 42 1193.7006/1193.737 +34.8/ +4.31235.7426 c12+ 2x42 1235.7111/1235.7475/1235.7839 +25.5/ -3.9/ -33.41253.7572 *c12+ 42 1253.7217/1253.7581 + 28.3/ -0.7• • • • •

9383.2622 c82+ 42 9383.2877/9383.3241 -2.7/ -6.610563.767 z94 10563.7159 +4.811548.262 z102 11548.324 -5.3

Mass Accuracy is Used to Distinguish between Isobaric Trimethylation and Acetylation

A single tri-methylation was on the N-terminus or on Lys-3. Lys-4 in both H2B.1 and H2B.2 can also be modified and the modification is acetylation. No C-terminal modification was observed.

ECD sequence coverage of the two isoforms

1APKKAPAAAA EKKVKKAPTT EKKNKKKRSE TFAIYIFKVL KQVHPDVGIS

51KKAMNIMNSF INDSFERIAL ESSKLVRFNK RRTLSSREVQ TAVKLLLPGE

101LARHAISEGT KAVTKFSSST N

1APKKAPAATT EKKVKKAPTT EKKNKKKRSE TFAIYIFKVL KQVHPDVGIS

51KKAMNIMNSF INDSFERIAL ESSKLVRFNK RRTLSSREVQ TAVKLLLPGE

101LARHAISEGT KAVTKFSSSS N

H2B.1

H2B.2

N-terminus or Lys-3 is trimethylated, Lys-4 is acetylated in both

CID Spectrum of an +84 Da modified H2B.1 Peptide

890.5119819.5279791.4913749.4550

722.4397720.4327677.3859

651.4087623.4134

606.3904

580.3822552.3975

509.3554

0

10

20

30

40

50

60

70

80

500 550 600 650 700 750 800 850 900 950

m /z

Ion

Cou

nts

m/z 490.31 (2+)b5

b8a8b7a7b6

a5

b9b4

481.2993387.7560

325.2264312.2248290.7023276.7004

232.1331186.1182

141.1077490.3065

431.7818

445.7797

410.2583

396.2588374.7353

360.7314339.2257

303.7079

240.1382

169.1013

161.0948

143.0843

126.0925

70.0676

115.0867

0

20

40

60

80

100

120

140

160

180

50 100 150 200 250 300 350 400 450 500

Ion

Cou

nts

P

y2326.2185

PAA

y3

b92+

b82+

a82+b72+

a72+MH2+

b62+a62+

a92+b52+

a2/AP-28

(a8-NH3)2+

AA-28

b2/PA

K (Ac)

K (Ac)

AAA-28

a52+ (MH-H2O)2+

APK*K* (Me3, Ac) APAAAA

Characteristic K(Ac) immonium ion 126.09 indicates the presence of Lys-acetylation

PK*K*AP

K*K*APAA-28

K*K*APAA

K*K*APAAA

PK*K*AP2+

[K*K*APAA-28]2+

K*K*APAA2+

Internal fragments show the modifications are on the two lysines, instead of the N-terminus.

APxxAPAAAA APwwAPAAAA APwx (or xw) APAAAA Massobs

(Da) Ions

Masscal

(Da) Error (ppm)

Ions

Masscal (Da)

Error (ppm)

Ions

Masscal (Da)

Error (ppm)

70.0676 P 70.0657 27

115.0867 AA-28 115.0871 -3

126.0925 K(Ac) 126.0919 27

141.1077 a2 141.1028 35

143.0843 AA 143.0821 15

161.0948 y2 161.0926 14

169.1013 b2 or PA 169.0977 21

232.1331 y3 232.1297 15

240.1382 PAA 240.1348 14

552.3975 a5 552.3510 84 a5 552.4237 -47 a5 552.3873 18 580.3822 b5 580.3459 63 b5 580.4186 -63 b5 580.3823 0 578.4262 PxxAP-28 578.3666 103 PwwAP-28 578.4394 -23 PwxAP-28 578.4030 40 580.3968 b5 580.3459 88 b5 580.4186 -38 b5 580.3823 25 606.4080 PxxAP 606.3615 77 PwwAP 606.4343 -43 PwxAP 606.3979 17 649.4450 a6 649.4037 64 a6 649.4765 -49 a6 649.4401 8 677.4436 b6 677.3986 66 b6 677.4714 -41 b6 677.4350 13 720.4550 a7 720.4408 20 a7 720.5136 -81 a7 720.4772 -31 748.4628 b7 748.4357 36 b7 748.5085 -61 b7 748.4721 -12 791.5098 a8 791.4779 40 a8 791.5507 -52 a8 791.5143 -6 819.5088 b8 819.4729 44 b8 819.5456 -45 b8 819.5092 0

Error for Common Ions (AVG ± STD) 20±9

Error for Modification Ions (AVG ± STD) 60±23 49±16 15±10

x—K(Ac); w– K(Me3)

Mass Accuracy of Fragment Ion Series Determines the +84 Da as a Tri-methylation and an Acetylation

Summary of H2B Posttranslational Characterization

Intact Protein Analysis Proteolytic Digest Analysis

Molecular Weight ECD- FT-ICR MS Trypsin Asp-N

H2B.1- Me3 (N*-or K3)-Me3 [1-45]- Me3 H2B.2- Me3 (N* or K3)-Me3 [1-45]- Me3

H2B.1- Me3 Ac (N* or K3)-Me3 K4-Ac

N*-Me3 K4-Ac and (K3K4)- Me3 Ac

(K3K4)-Me3 Ac Most

Abundant

H2B.2- Me3 Ac (N* or K3)-Me3 K4-Ac

N*-Me3 K4-Ac and (K3K4)-Me3 Ac

(K3K4)-Me3 Ac

H2B.1 56 Da (K3K4)- Me Ac H2B.1 70 Da N*-Me2 K4-Ac (K3K4)- Me2 Ac H2B.2 56 Da (K3K4)- Me Ac H2B.2 70 Da (K3K4)- Me2 Ac H2B.1- Me [1-45] Me H2B.1- Me2 [1-45] Me2 H2B.2- Me [1-45] Me

Less Abundant

H2B.2- Me2 [1-45] Me2 H2B.1/H2B.2-K41-Ac H2B.1 K111-Me2-3

Least Abundant

H2B.2 K111-Me3/Ac N* indicate N-terminus of the protein

Modified peptides in the tryptic digestIon detected MH+ calculated Structure [ppm]

418.608 (3+) 1253.758 4K(Ac)APAAAAEKKVK15 +40

438.611 (3+) 1313.779 4K(Ac)APAATTEKKVK15 +29

341.978 (4+) 1364.889 APKK (Ac,Me3)APAAAAEKK13 +44

413.773 (4+) 1652.011 APKK(Ac,Me3)APAATTEKKVK15 +35

Me3APKK(Ac)APAATTEKKVK15

410.269 (4+) 1637.995 Me2APKK(Ac) APAATTEKKVK15 +35

398.770 (4+) 1591.990 APKK(Ac,Me3)APAAAAEKKVK15

Me3APKK (Ac) APAAAAEKKVK15 +42

395.266 (4+) 1577.974 Me2APKK (Ac) APAAAAEKKVK15 +42

487.961 (3+) 1461.843 39VLK(Ac)QVHPDVGISK51 +17

631.668 (3+) 1892.972 HAISEGTK(Me2)AVTKFSSSTN121 +9

104HAISEGTKAVTK(Me3)FSSSSN121

1892.935 104HAISEGTKAVTK(Ac)FSSSSN121 +28

636.323(3+) 1906.950 104HAISEGTK(Me3)AVTKFSSSTN121 +2

Me3 @ N-terminus, Lys-3, Lys-111; Ac @ Lys-4; Lys-41[1-15] peptide always doubly modified!

Why to use both?

“bottom-up” approach: • More sensitive ~300 fmoles injected• Reveals more modificationsBUT• Misses the major protein component!

“top-down” reveals the relative distribution of differently modified populations

Conclusions

•Mass spectrometry is a sensitive, non-biased approach to peptide/protein modification analysis.

•Isolation, MS analysis has to be adjusted to the PTM of interest•Enrichment methods work much better on large amounts of protein than small.•For single protein/simple mixture PTM analysis, best approach is to use no enrichment, try to fragment as many components as possible then try to find spectra of modified peptides.

• Peptide-level analysis may not reflect accurately the composition of protein populations