1. ETD & ETD/PTR Electron Transfer Dissociation Proton Transfer Reaction
Title of sales presentation – Remarks: „Bruker Confidential“ needs to be erased (slide master!) for external use.
Electron Transfer Dissociation
Proton Transfer Reaction
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2. ETD versus CID 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
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 y2 y3 b3 b2 b1 y1 z3 z2 z1 c1 c2 c3
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3. Multiply charged analyte (n≥ 2)
ETD Reaction Scheme
Multiply charged analyte (n≥ 2)
Reagent radical anion
odd-electron
protonated
peptide n+
(n-1)+ - +
Electron-
transfer
Cleavage of
N-Cα bond
Prerequisite: multiply charged precursor ions, n ≥ 2 !
ETD is not applicable to 1+ or negatively charged ions
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4. 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.
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5. 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)
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6. detailed characterization: preparation artefacts
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
detailed characterization:
PTM
protein termini
mixed modifications
Protein ID is the first step in proteomics but in order to address the relevant topics a more detailed protein characterization is required.
PTM analysis is an important subject because the biological activity of proteins is often determined by post-translational modifications, e.g. phosphorylation, glycosylation and so on.
In the context of detailed protein characterization one needs to be able to handle multiple modifications as well as unexpected modifications. These modifications can have a natural origin or can be also preparation artefacts.
For the quality control of recombinant proteins the determination of N- and C-terminus are often of great importance.
Classical CID bottom-up approaches mostly fail to answer these questions whereas ETD and ETD/PTR are the perfect tools for these applications.
preparation artefacts
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7. + Strategy for phosphopeptides: PTMScanTM
PTMScanTM = neutral loss triggered ETD MS
Loss of H3PO4: m = 98
Combination of fast MS/MS for best sequence coverage (CID) and detailed analysis of modified peptides (ETD)
CID autoMS/MS
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) +
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8. PTMScanTM = Neutral Loss Triggered ETD
phosphopeptide from asialo fetuin (tryptic digest) 3+
760.6
355.1 1+
445.1
536.8 2+
644.3
738.4
880.9
956.4
1141.0
1287.1
303.2
440.3
844.4
932.4
1070.5
1166.7
0.0
0.5
1.0
1.5
2.0
2.5 2 4 6
200
400
600
800
1000
1200
1400
1600
m/z
728.0 3+
722.0
Intens.
x106
x104 MS 3+
760.6
728.0 3+
Auto CID MS/MS
loss of 32.6
triggers
ETD MS/MS of (3+)
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9. Phosphoscan CID versus PTMScan ETD
HTFSGVASVESSSGEAFHVGK, 2x phosphorylated, MW = Da
from asialo fetuin (tryptic digest)
CID: merged MS2 & pseudoMS3
CID MSn:
Phosphorylation can not be assigned
Question 2: Why is everybody so excited about this? What is the motivation to use ETD and what are main applications
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10. Phosphoscan CID versus PTMScan ETD
HTFSGVASVES*SS*GEAFHVGK, 2x phosphorylated, MW = Da
from asialo fetuin (tryptic digest)
ETD MS²
ETD ► Phosporylation at S11 and S13
Question 2: Why is everybody so excited about this? What is the motivation to use ETD and what are main applications
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11. ? 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
330.6 2+
444.2
660.2 1+
826.3
1134.7
1331.5
1495.2
551.2 3+
MS, 11.7 min
516.3
CID (551.2)
259.1
361.2
798.3
918.3
1020.5
1146.4
1293.4
1595.7
ETD (551.2)
0.25
0.50
0.75 8
x10
Intens. 2 4 7
0.0
0.5
1.0
1.5
2.0 6
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 ! ?
The first example deals with the identification of phosphorylation sites from different caseins. For this purpose the digested protein mixture was analyzed by alternating CID-ETD.
The proteins and the phosphorylation sites were identified via Mascot database search. During the result evaluation it became obvious that several good quality ETD spectra remained unassigned. One of these spectra is shown here. The corresponding CID spectrum is shown as well, it contains only one fragment deriving from a neutral loss of 105 Da which differs quite a lot from the expected neutral loss of 98 corresponding to phosphoric acid.
So the question is, what causes a neutral loss of 105 and prevents the ETD spectra from identification.
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12. What causes a Neutral Loss of 105 Da ?
A neutral loss of 105 Da can occur from carbamidomethylated methionine:1)
carbamidomethylated
methionine
Loss of 105 Da
A neutral loss of 105 Da is already described in the literature. It occurs from carbamidomethylated methionine. The picture shows the formation of the neutral loss fragment from the carbamidomethylated methionine during the CID process.
The carbamidomethylation of methionine is a sample preparation artefact. It can be formed as side product during cysteine alkylation.
Carbamidomethylation of methionine is a sample preparation artefact.
It can be formed as side product during cysteine alkylation.
1) Krüger et al., Rapid Commun. Mass Spectrom. 2005; 19:
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13. NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK
Mascot Database Search Results for α-S2-Casein
Comparison of search results without and with modification Carbamidomethyl (M)
without
with modification
Carbamidomethyl (M)
The additional modification Carbamidomethyl (M) can be included in the Mascot search parameter. Here, the search results for α-s2-casein with different search parameters are compared. The second search result was obtained considering also methionine carbamidomethylation, the first one without.
Considering this modification results in the identification of two more phosphopeptides based on their ETD spectra.
► With the knowledge of camMet as sample preparation artefact, two additional phosphopeptides are identified via ETD
NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK
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14. ETD Spectrum of TVDcamMEpSTEVFTKK
ETD of (3+), tR = 11.8 min M* S* S*
... Annotation clearly confirms the presence of phosphorylated serine as well as the carbamidomethylated Methione
The annotated ETD spectrum of one of the two phosphopeptides clearly shows the presence of both labile modifications: the serine phosphorylation as well as the methionine carbamidomethylation.
The CID spectrum did not give an identification due to the absence of any fragments from the peptide backbone.
► A single ETD spectrum allows for the identification of phosphorylation sites also in the presence of other labile modifications.
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15. Strategy for glycopeptide analysis
CID autoMS/MS analysis of the digested glycoprotein in enhanced resolution mode
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
ETD experiment, either in autoMSn mode with or w/o inclusion list or in manual MS/MS mode to obtain best data quality.
Define the glycan moiety as modification in BioTools and match the ETD spectrum with the modified known sequence.
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16. Glycopeptide analysis using CID
Fragments come almost exclusively from the cleavage of glycan moiety
IgG3 tryptic digest
glycopeptide MW 2602 Da
366.1
528.2
690.3
893.3
944.9
1025.6
1046.5
1098.9
1157.6
1200.2
1229.0
1360.7
1470.7
1506.7
1563.7
1709.8
1887.8
2400.0
400
800
1200
1600
2000
2400
pep *
m/z
N-acetylglucosamine
galactose
mannose
fucose
sialic acid
pep
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17. Side chain cleavage of N-glyc Asn
Glycopeptide analysis using ETD
Fragments arise from the cleavage of peptide backbone
IgG3 tryptic digest NH
C=O
CH3
Asn
2603.2
2054.9
2201.9
2330.0
2403.1
2458.0
2560.1
1200
1600
2000
2400
495.2
516.3
687.5
708.4
927.4
1041.4
1099.4
400
800
408.2
z3.
z4.
z9.
2587.1
z8.
z7.
z6.
z5.
[M+2H]2+
1301.6
x 5
Side chain cleavage of N-glyc Asn
m/z
Glu-Gln-Gln-Phe-Asn-Ser-Thr-Phe-Arg z4 z5 z6 z7 z8 z9 z3
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18. ETD CID Glycopeptide analysis using CID and ETD EQQFNSTFR
CID and ETD provide complementary information for glycopeptide identification
Peptide Sequence
Glycan moiety
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19. multiply charged fragment ions up to z=4 are identified
ETD analysis of large peptides
galanin-like peptid (GALP) MWmono = Da
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
c32
(z+1) 31
(z+1) 1
c 1
150
160
170
180
190
m/z 3+
(z+1) 50 2+
(z+1) 33
1696
1700
1704
m/z
0.0
0.5
1.0
1.5
2.0 5
x10
Intens.
500
1000
1500
2000
2500
m/z 2+
(z+1) 54
2780
2784
2788
m/z
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20. ETD of large peptides galanin-like peptid (GALP) MWmono = 6200.3 Da
Deconvoluted spectrum
500
1000
1500
2000
2500
3000 c H R G W T L N S A Y P V
z+1 K E
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
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
m/z
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21. detailed characterization: preparation artefacts
Use of ETD for detailed Protein Characterization
protein ID
detailed characterization:
PTM
protein termini
ETD-PTR top-down analysis for the determination of N- & C-termini of intact proteins
mixed modifications
Protein ID is the first step in proteomics but in order to address the relevant topics a more detailed protein characterization is required.
PTM analysis is an important subject because the biological activity of proteins is often determined by post-translational modifications, e.g. phosphorylation, glycosylation and so on.
In the context of detailed protein characterization one needs to be able to handle multiple modifications as well as unexpected modifications. These modifications can have a natural origin or can be also preparation artefacts.
For the quality control of recombinant proteins the determination of N- and C-terminus are often of great importance.
Classical CID bottom-up approaches mostly fail to answer these questions whereas ETD and ETD/PTR are the perfect tools for these applications.
preparation artefacts
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22. Precusor Isolation [M+12H]12+
ETD & PTR for large peptides / small proteins
Ubiquitin, bovine ( 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
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23. ETD & PTR for large peptides / small proteins
Ubiquitin, bovine (MW = Da)
500
1000
1500
2000
2500
m/z
ETD - PTR
fragment charge states ≤ 6+
Maximum Resolution Mode
PTR Proton Transfer Reaction
500
1000
1500
2000
2500
m/z
12+
11+
10+ 9+
ETD
fragment charge states ≤ 12+
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24. + + - - 12+ Principle of ETD-PTR Top Down Analysis
ETD ► Production of highly charged fragment ions from intact proteins + n+
Electron
Transfer
12+ -
multiply charged fragment ions n =11, 10, 9, 8, ...
fragment ions with reduced charge states m = 6, 5, 4, 3, 2, 1 +
Proton
Transfer - n+ m+
PTR ► Charge reduction using Proton Transfer Reaction
Only few fragment ion are isotopically resolved as the major amount of the fragment ions are highly charged
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25. Bruker PTR reagent from fluoranthene
PTR-reagents O -
Benzoate anion (Hunt, Coon et al.)  need two separate reagent reservoirs for ETD and PTR
Perfluoro-1,3-dimethylcyclohexane = PDCH (McLuckey et al.) C - H
Bruker PTR reagent from fluoranthene - •
+ H
C16H10• -
C16H11-
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26. Ubiquitin, bovine (MW = 8559.6 Da)
ETD & PTR for large peptides / small proteins
Deconvoluted spectrum
Ubiquitin, bovine (MW = 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)
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