Page 1 ETD & ETD/PTR Electron Transfer Dissociation Proton Transfer Reaction.

Page 1

ETD & ETD/PTR

Electron Transfer DissociationProton Transfer Reaction

Page 2

Conventional (resonant) CID

• via several collisions with Helium

precursor ion is internally heated• preferences for weak bond cleavages• nearby selected amino acids (E, D, P)

backbone cleavage is preferred• b- and y-ions (and internal fragments)• best fragment spectra from 2+ ions

ETD

• electron transfer surpasses internal heating• rapid bond cleavage (no energy dissipation) • random fragmentation of peptide backbone• leaves labile bonds like from PTMs intact

• N-C bond cleavage yields c- and z-ion• preferable charge state z > 2

ETD versus CID

y2y3

b3b2b1

y1z3 z2 z1

c1 c2 c3

Page 3

ETD Reaction Scheme

odd-electronprotonated

peptide

Multiply

charged

analyte

(n≥ 2)

Electron-transfer

Cleavage ofN-Cα bondn+ + (n-1)+-

Prerequisite: multiply charged precursor ions, n ≥ 2 !

ETD is not applicable to 1+ or negatively charged

ions

Reagent

radical

anion

Page 4

Even though the N-C bond is cleaved no respective c and z fragments

are formed since they stay connected via the Proline ring system.

ETD: No Cleavage at Proline

Page 5Page 5

The “3D Advantage”

Cations and anions are pushed

towards the center of the trap

Direct ETD reaction as soon as

anions enter the trap

Better cross sections for ion-ion-

reactions in 3D trap due to

compression into the same

globular volume

highly efficient ETD reaction

Non-linear Paul Trap:

Dual injection and storage of ions of both

polarities peptide cations & reagent anions

Spec: ≥ 18 unique peptides from 5 fmol BSA on column (Easy-nLC)

Page 6Page 6

Use of ETD for detailed Protein Characterization

• Analysis of post-translational

modifications (PTMs)

• phosphorylation

• glycosylation

• deamidation etc.

• Identification of sample preparation

artifacts

• MS/MS of large peptides

• Combination of CID and ETD data

for improved characterization of

peptides and proteins, e.g. for QC

applications.

protein ID

PTM

mixed modifications

proteintermini

preparation artefacts

detailed characterization:

Page 7Page 7

Strategy for phosphopeptides: PTMScanTM

PTMScanTM = neutral loss triggered ETD

+

Loss of Δm/z 49, 32.6

and product ion among

top N most intense

MS/MS fragments ?No !

Yes !

ETD auto-MS2 of

original intact

PRECURSOR ion

CID autoMS3 of

neutral loss

product ion(s)

CID autoMS/MS

MS Loss of H3PO4: m = 98

Combination of

fast MS/MS for best

sequence coverage

(CID)

and

detailed analysis of

modified peptides

(ETD)

Page 8Page 8

355.11+

445.11+

536.82+

644.32+

738.42+

880.92+

956.42+

1141.02+

1287.1

303.21+ 440.3

1+

844.41+

932.41+

1070.51+

1166.71+

0.0

0.5

1.0

1.5

2.0

2.5

0

2

4

6

200 400 600 800 1000 1200 1400 1600 m/z

728.03+

722.03+

Intens.x106

Intens.x104

loss of 32.6

triggersETD MS/MS

of 760.6 (3+)

MS

Auto CID MS/MS

728.0

3+

3+

760.6

phosphopeptide from asialo fetuin (tryptic digest)

3+760.6

PTMScanTM = Neutral Loss Triggered ETD

Page 9Page 9

CID MSn:Phosphorylation can not be assigned

HTFSGVASVESSSGEAFHVGK, 2x phosphorylated, MW = 2279.9 Da

from asialo fetuin (tryptic digest)CID: merged MS2 & pseudoMS3

Phosphoscan CID versus PTMScan ETD

Page 10Page 10

HTFSGVASVES*SS*GEAFHVGK, 2x phosphorylated, MW = 2279.9 Da

from asialo fetuin (tryptic digest)ETD MS²

ETD ► Phosporylation at S11 and S13

Phosphoscan CID versus PTMScan ETD

Page 11Page 11

Identification of phosphorylation sites from a mixture of different caseins.

► Observation of several CID spectra showing a neutral loss of 105 Da instead

of 98!

Those spectra could not be identified via Mascot database search

?330.6

2+ 444.22+ 660.2

1+

826.32+

1134.7 1331.5 1495.2

551.23+

551.23+

MS, 11.7 min

516.33+CID (551.2)

259.11+

361.21+

551.23+

798.32+

918.31+

1020.51+

1146.41+

1293.41+ 1595.7

1+

ETD (551.2)

0.25

0.50

0.75

8x10Intens.

2

4

7x10

0.0

0.5

1.0

1.5

2.06x10

200 400 600 800 1000 1200 1400 1600 m/z

ETD : Good fragment pattern !

∆m = - 35 → Neutral Loss of 105 Da

CID : Almost no b- and y-ions !

Alternating CID-ETD for phosphopeptide analysis

Page 12Page 12

What causes a Neutral Loss of 105 Da ?

A neutral loss of 105 Da can occur from carbamidomethylated

methionine:1)

Loss of 105 Dacarbamidomethylated

methionine

1) Krüger et al., Rapid Commun. Mass Spectrom. 2005; 19: 1709-1716.

Carbamidomethylation of methionine is a sample preparation

artefact.

It can be formed as side product during cysteine alkylation.

Page 13Page 13

Mascot Database Search Results for α-S2-Casein

Comparison of search results without and with modification Carbamidomethyl (M)

► With the knowledge of camMet as sample preparation artefact,

two additional phosphopeptides are identified via ETD

NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK

with modification

Carbamidomethyl (M)

without

Page 14Page 14

ETD Spectrum of TVDcamMEpSTEVFTKK

ETD of 551.2 (3+), tR = 11.8 min

► A single ETD spectrum allows for the identification of

phosphorylation sites also in the presence of other labile

modifications.

M* S*S*

Page 15Page 15

Strategy for glycopeptide analysis

1. CID autoMS/MS analysis of the digested glycoprotein in enhanced

resolution mode

2. Identification of the glycopeptides:

• check for the presence of typical CID marker ions:

- HexNAc: m/z 204

- HexNAcHex: m/z 366

- NeuAc: m/z 292, 274, 256

- HexHexNAcNeuAc: m/z 657

• only for O-glycans: check for neutral loss chromatograms, e.g. for

hexose (54, 81, 162), HexNAc (101.5, 203), NeuAc (145.5, 291)

• annotation of the sugar distances in order to determine the glycan

residue

3. ETD experiment, either in autoMSn mode with or w/o inclusion list or in

manual MS/MS mode to obtain best data quality.

4. Define the glycan moiety as modification in BioTools and match the ETD

spectrum with the modified known sequence.

Page 16Page 16

pep

36

6.1

52

8.2

69

0.3

89

3.3

94

4.9

10

25

.61

04

6.5

10

98

.9

11

57

.61

20

0.2

12

29

.0

13

60

.7

14

70

.71

50

6.7

15

63

.7

17

09

.8

18

87

.8

24

00

.0

400 800 1200 1600 2000 2400

pep

pep

pep

pep

pep

pep

pep

pep

pep

pep

pep

**

**

m/z

Fragments come almost exclusively from the cleavage of glycan moiety

IgG3 tryptic digest

glycopeptide

MW 2602 Da

N-acetylglucosaminegalactosemannosefucosesialic acid

Glycopeptide analysis using CID

Page 17Page 17

Fragments arise from the cleavage of peptide backbone

Glycopeptide analysis using ETD

NHC=OCH3

Asn

26

03

.2

20

54

.9

22

01

.9

23

30

.0

24

03

.1

24

58

.02

56

0.1

1200 1600 2000 2400

49

5.2

51

6.3

68

7.5

70

8.4

92

7.4

10

41

.41

09

9.4

400 800

40

8.2

z3.

z4.

z9.

25

87

.1

z8.

z7.

z6.

z5.

[M+2H]2+

13

01

.6

x 5

Side chain cleavage of N-glyc Asn

m/z

Glu-Gln-Gln-Phe-Asn-Ser-Thr-Phe-Arg

z4z5z6z7z8z9 z3

IgG3 tryptic digest

Page 18Page 18

Glycopeptide analysis using CID and ETD

CID and ETD provide complementary information for glycopeptide

identification

Peptide Sequence

Glycan moiety

EQQFNSTFR

ETD

CID

Page 19Page 19

0.0

0.5

1.0

1.5

2.0

5x10

Intens.

500 1000 1500 2000 2500 m/z

galanin-like peptid (GALP)

MWmono = 6200.3 Da

(z+1) 1

c 1

150 160 170 180 190 m/z

multiply charged fragment ions up to

z=4 are identified

(Enhanced scan mode,

8100 m/z per sec)3+c23

800 805 810 815 m/z

4+c31

2+c16

4+c32

4+(z+1) 31

3+(z+1) 50

2+(z+1) 33

1696 1700 1704 m/z

2+(z+1) 54

2780 2784 2788 m/z

ETD analysis of large peptides

Page 20Page 20

galanin-like peptid (GALP)

MWmono = 6200.3 Da

ETD of large peptides

Deconvoluted spectrum

m/z500 1000 1500 2000 2500 3000

c H R G R GG W T L N SAG Y GP V L P S R GGz+1 L AT KGKGGGE A

c 3

c 4

c 5

c 6

c 7

c 8

c 9 c 10

c 11

c 12

c 13

c 14

c 15

z+1 15

c 16

c 17

c 19

z+1 19

c 20

c 21

z+1 21

c 22

z+1 22

c 23

z+1 23

z+1 24

c 25

z+1 25

c 26

z+1 26

c 27

z+1 27

c 28

z+1 28

z+1 29

c 30

z+1 30

c 31

z+1 31

z+1 32

3500 4000 4500 5000 5500 6000

GKGK T A LGR S H L L L Y S N L T W GG R H V P

c 32

c 33

z+1 33

c 34

z+1 34

c 35 c 36

z+1 36

c 37

z+1 37

c 38

z+1 38

c 39

c 40

z+1 41

z+1 42

c 43

z+1 43

z+1 44

c 45

z+1 46

z+1 47

c 48

z+1 48

z+1 49

c 50

z+1 50

z+1 51

z+1 52

z+1 53

z+1 54

z+1 56

c 57

z+1 57

z+1 58

z+1 59

c 60

Page 21Page 21

Use of ETD for detailed Protein Characterization

• ETD-PTR top-down analysis for the

determination of N- & C-termini of

intact proteins

protein ID

PTM

mixed modifications

proteintermini

preparation artefacts

detailed characterization:

Page 22Page 22

ETD & PTR for large peptides / small proteins

Ubiquitin, bovine (8559.6 Da)

500 1000 1500 2000 2500 m/z

12+

13+ 11+

10+9+

MS

500 1000 1500 2000 2500 m/z

Precusor Isolation

[M+12H]12+

12+

500 1000 1500 2000 2500 m/z

12+

11+

10+

9+

ETD

Page 23Page 23

Ubiquitin, bovine (MW = 8559.6 Da)

500 1000 1500 2000 2500 m/z

12+

11+

10+

9+

ETDPTR

Proton

Transfer

Reaction

fragment charge states ≤ 12+

500 1000 1500 2000 2500 m/z

ETD - PTR

fragment

charge states

≤ 6+

ETD & PTR for large peptides / small proteins

Maximum Resolution Mode

Page 24Page 24

Principle of ETD-PTR Top Down Analysis

multiply charged fragment ions n =11, 10, 9, 8, ...

+ n+ n+n+ n+Electron

Transfer12+

-

ETD ► Production of highly charged fragment ions from intact proteins

fragment ions with reduced charge states m = 6, 5, 4, 3,

2, 1

+ Proton

Transfer-n+ m+m+ m+

PTR ► Charge reduction using Proton Transfer Reaction

Page 25Page 25

PTR-reagents

Bruker

PTR reagent from

fluoranthene

+ H

Benzoate anion (Hunt, Coon et al.)

need two separate reagent reservoirs for ETD

and PTR

Perfluoro-1,3-dimethylcyclohexane = PDCH (McLuckey et al.)

-•

C16H10•

- C16H11-

C-

H

O-

O

Page 26Page 26

Deconvoluted spectrum

ETD & PTR for large peptides / small proteins

Ubiquitin, bovine (MW = 8559.6 Da)

Applications: e.g. QC of recombinant proteins, isolated proteins e.g. from cell lysates

Advantages: no 1/3 cut-off, PTMs visible, good sequence coverage, N/C-termini included!

Limitations: slow for LC separations, off-line techniques may be required (direct infusion, off-line

nanospray,

e.g. NanomateTM)