ETD & ETD/PTR
Electron Transfer Dissociation
Proton Transfer Reaction
Page 1
ETD versus CID
ETD
Conventional (resonant) CID
• electron transfer surpasses internal heating
• rapid bond cleavage (no energy dissipation)
• random fragmentation of peptide backbone
• leaves labile bonds like from PTMs intact
• 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
• N-C bond cleavage yields c- and z-ion
• preferable charge state z > 2
y3 z3
b 1 c1
Page 2
y2 z2
b 2 c2
y1 z1
b 3 c3
ETD Reaction Scheme
Multiply
charged
Reagent
radical
anion
analyte
(n≥ 2)
n+
+
-
odd-electron
protonated
peptide
Electrontransfer
(n-1)+
Prerequisite: multiply charged precursor ions, n ≥ 2 !
ETD is not applicable to 1+ or negatively charged ions
Page 3
Cleavage of
N-Cα bond
ETD: No Cleavage at Proline
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.
Page 4
The “3D Advantage”
Non-linear Paul Trap:
Dual injection and storage of ions of both polarities
peptide cations & reagent anions
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
Spec: ≥ 18 unique peptides from 5 fmol BSA on column (Easy-nLC)
Page 5
Use of ETD for detailed Protein Characterization
•
Analysis of post-translational
modifications (PTMs)
• phosphorylation
protein ID
• glycosylation
detailed characterization:
PTM
mixed
modifications
protein
termini
preparation
artefacts
Page 6
• 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.
Strategy for phosphopeptides: PTMScanTM
PTMScanTM = neutral loss triggered ETD
MS
Loss of H3PO4: m = 98
CID autoMS/MS
Combination of
fast MS/MS for best
sequence coverage (CID)
Loss of Δm/z 49, 32.6
and
and product ion among
top N most intense
No !
MS/MS fragments ?
detailed analysis of
modified peptides (ETD)
Yes !
ETD auto-MS2 of
original intact
PRECURSOR ion
Page 7
+
CID autoMS3 of
neutral loss
product ion(s)
PTMScanTM = Neutral Loss Triggered ETD
phosphopeptide from asialo fetuin (tryptic digest)
Intens.
x106
2.5
3+
760.6
2+
738.4
2+
880.9
MS
2.0
1.5
2+
536.8
1.0
3+
728.0
0.5
2+
956.4
2+
644.3
3+
760.6
2+
1141.0
1+
1+
445.1
355.1
1287.1
0.0
Intens.
x104
Auto CID MS/MS
3+
722.0
6
loss of 32.6
3+
728.0
4
2
1+
303.2
1+
440.3
1+
1+
844.4 932.4
1+
1+
1070.5 1166.7
0
200
Page 8
400
600
800
1000
1200
triggers
ETD MS/MS
of 1400
760.6 (3+)
1600 m/z
Phosphoscan CID versus PTMScan ETD
HTFSGVASVESSSGEAFHVGK, 2x phosphorylated, MW = 2279.9 Da
from asialo fetuin (tryptic digest)
CID: merged MS2 & pseudoMS3
CID MSn:
Phosphorylation can not be assigned
Page 9
Phosphoscan CID versus PTMScan ETD
HTFSGVASVES*SS*GEAFHVGK, 2x phosphorylated, MW = 2279.9 Da
from asialo fetuin (tryptic digest)
ETD MS²
ETD ► Phosporylation at S11 and S13
Page 10
Alternating CID-ETD for phosphopeptide analysis
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
Intens.
x108
MS, 11.7 min
2+
2+
444.2
330.6
0.75
0.50
1+
3+ 660.2
551.2
2+
826.3
0.25
x107
4
1134.7
CID (551.2)
1495.2
∆m = - 35 → Neutral Loss of 105 Da
3+
516.3
2
x106
2.0
1331.5
CID : Almost no b- and y-ions !
3+
551.2
ETD (551.2)
1.5
2+
798.3
ETD : Good fragment pattern !
1.0
0.5
1+
1+
1+
918.3 1020.5 1146.4
1+
1+
259.1 361.2
0.0
200
Page 11
400
600
800
1000
1200
1+
1595.7
1+
1293.4
1400
1600
m/z
?
What causes a Neutral Loss of 105 Da ?
A neutral loss of 105 Da can occur from carbamidomethylated methionine:1)
carbamidomethylated
Loss of 105 Da
methionine
Carbamidomethylation of methionine is a sample preparation artefact.
It can be formed as side product during cysteine alkylation.
1) Krüger
Page 12
et al., Rapid Commun. Mass Spectrom. 2005; 19: 1709-1716.
Mascot Database Search Results for α-S2-Casein
Comparison of search results without and with modification Carbamidomethyl (M)
without
with modification
Carbamidomethyl (M)
► With the knowledge of camMet as sample preparation artefact,
two additional phosphopeptides are identified via ETD
NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK
Page 13
ETD Spectrum of TVDcamMEpSTEVFTKK
ETD of 551.2 (3+), tR = 11.8 min
M*
S*
S*
► A single ETD spectrum allows for the identification of phosphorylation
sites also in the presence of other labile modifications.
Page 14
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 15
Glycopeptide analysis using CID
Fragments come almost exclusively from the cleavage of glycan moiety
pep
pep
pep
1887.8
pep
2400.0
1709.8
1506.7
pep
1200
*
*
1470.7
1157.6
pep
*
*
1563.7
1098.9
1046.5
800
pep
pep
944.9
1025.6
893.3
690.3
528.2
366.1
400
1200.2
pep
pep
Page 16
1229.0
pep
N-acetylglucosamine
galactose
mannose
fucose
sialic acid
pep
1360.7
IgG3 tryptic digest
glycopeptide
MW 2602 Da
1600
2000
2400 m/z
Glycopeptide analysis using ETD
Fragments arise from the cleavage of peptide backbone
z8
z7
z6
z5
z4
z3
[M+2H]2+
2603.2
z9
1301.6
IgG3 tryptic digest
Side chain cleavage of N-glyc Asn
Glu-Gln-Gln-Phe-Asn-Ser-Thr-Phe-Arg
CH3
C=O
NH
2560.1 2587.1
z9.
x5
Page 17
1099.4
800
z8.
1600
2000
2458.0
2403.1
2330.0
2054.9
1200
2201.9
.
z6. z7
1041.4
927.4
687.5
708.4
495.2
516.3
400
z5.
Asn
408.2
z3.
z4.
2400 m/z
Glycopeptide analysis using CID and ETD
CID and ETD provide complementary information for glycopeptide identification
Peptide Sequence
ETD
EQQFNSTFR
Glycan moiety
CID
Page 18
ETD analysis of large peptides
galanin-like peptid (GALP)
MWmono = 6200.3 Da
multiply charged fragment ions up to
z=4 are identified
(Enhanced scan mode,
3+
c23
8100 m/z per sec)
4+
c31
c1
(z+1) 1
Intens.
5
x10
800
2+
c16
4+
(z+1) 31
805
810
160
170
180
190
m/z
815
3+
(z+1) 50
2+
(z+1) 33
150
4+
c32
m/z
2.0
1696
1700
1704
m/z
1.5
2+
(z+1) 54
1.0
0.5
0.0
Page 19
2780
500
1000
1500
2000
2784
2500
2788
m/z
m/z
ETD of large peptides
galanin-like peptid (GALP)
MWmono = 6200.3 Da
Deconvoluted spectrum
c
z+1
H R G R GG W T L N SAG Y
GP V L
PS R
GGG K GK T A LG
H L
L AT KGKGGGE A R S
L L Y
S N L T W GG R
H VP
z+1 59
z+1 23
c 38
c 23
z+1 37
z+1 22
c 60
z+1 32
z+1 31
z+1 29
c 33
c 31
c 32
z+1 27
c 30
c 27
c 20
z+1 28
z+1 19
c 19
z+1 26 z+1 30
z+1 15
c6
c 13
c8
c 12
c4
c3
c5
c 11
c7
c 9 c 10
c 16
c 15
z+1 33
c 22
c 26
z+1 21
z+1 25
c 21
z+1 53
z+1 52
z+1 50
z+1 36
z+1 34
c 28
z+1 24
c 14
Page 20
1000
z+1 58
z+1 57
z+1 46
c 48
z+1 43c 45
c 43 z+1 44
z+1 38
z+1 42
c 25
1500
z+1 41
c
c 37 c 40
35 c 36
c 34
c 39
c 17
500
z+1 48 z+1 51
z+1 47 c 50
z+1 56
z+1 54
c 57
z+1 49
2000
2500
3000
3500
4000
4500
5000
5500
6000 m/z
Use of ETD for detailed Protein Characterization
protein ID
detailed characterization:
PTM
mixed
modifications
protein
termini
preparation
artefacts
Page 21
•
ETD-PTR top-down analysis for the
determination of N- & C-termini of
intact proteins
ETD & PTR for large peptides / small proteins
Ubiquitin, bovine (8559.6 Da)
MS
12+
13+ 11+
10+
9+
500
1000
12+
1500
2000
2500
Precusor Isolation
[M+12H]12+
m/z
ETD
11+
12+
500
1000
1500
2000 10+2500
m/z
9+
500
Page 22
1000
1500
2000
2500
m/z
ETD & PTR for large peptides / small proteins
Ubiquitin, bovine (MW = 8559.6 Da)
ETD - PTR
Maximum Resolution Mode
fragment charge
states ≤ 6+
500
PTR
Proton
Transfer
Reaction
1000
1500
2000
ETD
11+
12+
10+
fragment charge
states ≤ 12+
9+
500
Page 23
m/z
2500
1000
1500
2000
2500
m/z
Principle of ETD-PTR Top Down Analysis
ETD ► Production of highly charged fragment ions from intact proteins
12+
+
-
Electron
Transfer
n+
n+
n+
n+
multiply charged fragment ions n =11, 10, 9, 8, ...
PTR ► Charge reduction using Proton Transfer Reaction
n+
+
-
Proton
Transfer
m+
m+
m+
fragment ions with reduced charge states m = 6, 5, 4, 3, 2, 1
Page 24
PTR-reagents
-
O
O
Benzoate anion (Hunt, Coon et al.)
 need two separate reagent reservoirs for ETD and PTR
Perfluoro-1,3-dimethylcyclohexane = PDCH (McLuckey et al.)
•-
+
H
H
-
C
C16H10• Page 25
C16H11-
Bruker
PTR reagent from
fluoranthene
ETD & PTR for large peptides / small proteins
Deconvoluted spectrum
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)
Page 26