CH 908: Mass Spectrometry Lecture 9 Electron Capture Dissociation of Peptides and Proteins

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CH 908 Mass SpectrometryLecture 9Electron
Capture Dissociation of Peptides and Proteins

• Prof. Peter B. OConnor

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Objectives for this lecture

• Odd vs. Even electron fragmentation reactions
• First paper
• History of the mechanisms
• hot hydrogen model
• UW model
• Odd results noticed with polymers and
  zinc-binding peptides
• Radical cascade mechanism
• cyclic peptide data
• radical migration
• double resonance
• radical traps
• Utility
• Disulphide bonds
• Protein structural studies
• Labile modifications
• Isomer determination
• HDX?
• Other ExD methods

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Odd vs. Even Electron Fragmentation

• Even electron proton rearrangements
• Odd electron radical rearrangements
• Non-ergodic fragmentation FAST!!

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ECD Spectrum
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Hot H Mechanism of ECD
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UW Mechanism
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Activated-Ion ECD
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Activated-Ion ECD
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Activated-Ion ECD
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Kjeldsen, F. Haselmann, K. F. Budnik, B. A.
Jensen, F. Zubarev, R. A. Dissociative capture
of hot (3-13 eV) electrons by polypeptide
polycations an efficient process accompanied by
secondary fragmentation Chem Phys. Lett. 2002,
356, 201-206.
Hot ECD
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Hot ECD
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The effect of metals In this case, Zinc binding
killed the electron thus killing fragmentation
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Electron capture dissociation of polyethylene
glycol HO-(CH2-CH2-O)n-H
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Cyclic peptide structures
cyclo-LLFHWAVGH
gramicidin S
cyclosporin A

• Two histidines
• Tryptophan
• phenylalanine

• Ornithine
• Proline
• Phenylalanine

• N-Methylated
• Two unusual
• Amino acids

Leymarie, N. Costello, C. E. O'Connor, P. B.
Electron capture dissociation initiates a free
radical reaction cascade J Am Chem Soc 2003, 125,
8949-8958.
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ECD of cyclo- LLFHWAVGH
X12
X30
(M2H)2
cleavage assignable to backbone cleavage cleavage
assignable to loss of H, H2O, NH3, CO,
CONH cleavage assignable to sidechain
fragmentation
?
?
?
?
?
?
?
? electronic noise, ? Harmonic
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• The free radical reaction cascade will continue
  until one of the following occurs
• The various energy losses in the reactions and in
  black body radiation cool the system sufficiently
  that further rearrangments are impossible
• The free radical is eliminated leaving the charge
  containing peptide in an even-electron state
• The free radical is stabilized at a site of low
  reactivity

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Is the Free Radical Cascade mechanism really
correct?

• FRC mechanism implies radical migration.
  Calculations suggest radical will migrate to the
  alpha carbon. If so, deuterium labeling those
  (non exchangeable) positions should cause the
  fragments to show D scrambling
• FRC mechanism implies that the radical
  intermediates are long lived ( microseconds or
  more). This can be tested by double resonance.
• FRC mechanism implies radical migration.
  Addition of a radical trap moiety to the peptide
  should radically change the fragmentation.

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Free Radical Cascade in Linear Peptide
ECDDeuterium scrambling by Hydrogen Abstraction
C
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ECD of D-labeled synthetic peptides H/D
Scrambling
BUSM 1 undeuterated BUSM 2 deuterated
RAG2DADG2DDADG2DDAG2DAAR
Top ECD of BUSM 1 Middle 0.2 eV ECD of BUSM
2 Bottom 9 eV ECD of BUSM 2 Spectra of BUSM 2
were shifted left by 2 Daltons per glycine to
align with those of BUSM 1
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H/D Scrambling Results

• The initial radical does migrate to a-carbons
• The best mechanism supports a hydrogen bonded
  dimer as the long-lived radical intermediate

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Is the Free Radical Cascade mechanism really
correct?

• FRC mechanism implies radical migration.
  Calculations suggest radical will migrate to the
  alpha carbon. If so, deuterium labeling those
  (non exchangeable) positions should cause the
  fragments to show D scrambling
• FRC mechanism implies that the radical
  intermediates are long lived ( microseconds or
  more). This can be tested by double resonance.
• FRC mechanism implies radical migration.
  Addition of a radical trap moiety to the peptide
  should radically change the fragmentation.

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Double Resonance
short lived

X

X

long lived


short lived
Resonantly Eject
Timeframe 0.01 1 msec
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Double Resonance
Which fragments come from long lived
intermediates?
Resonant Excite
Fragments derived from long-lived intermediates
will disappear from spectra Fragments from
short-lived intermediates will not change
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Substance P ECD RPKPQQFFGLM-NH2
M2
c5
c10
c7
M2H
c6
c4
c8
c9

a7
z9
w2
m/z
m/z
electronic noise
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Substance P
S
O
N
H
H
N
2
N
H
2
N
H
N
H
H
O
N
H
O
H
N
O
2
O
O
N
H
2
N
H
O
N
H
c4
N
N
z9
H
H
N
N
N
N
O
O
O
H
H
O
O
N
H

3
N
H
O
2
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BUSM 1 ECD RAAA GADG DGAG ADAR
c6
c8
c5
c3
a7
a6
a5
b7
y7
w2
c4
a4
c7
z7
z6
a7
m/z
-34
-CO
-45
c15
-NH3
-60
c12
-104/5
c13
c10
c14
-101
M2H
z12
y9
z14
z9
-149
-72
c11
a13
b14
a15
b9
-78
a11
a12
c9
a14
z13
z15
z8
a9
a10
-130
m/z
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Is the Free Radical Cascade mechanism really
correct?

• FRC mechanism implies radical migration.
  Calculations suggest radical will migrate to the
  alpha carbon. If so, deuterium labeling those
  (non exchangeable) positions should cause the
  fragments to show D scrambling
• FRC mechanism implies that the radical
  intermediates are long lived ( microseconds or
  more). This can be tested by double resonance.
• FRC mechanism implies radical migration.
  Addition of a radical trap moiety to the peptide
  should radically change the fragmentation.

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Radical traps
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Radical traps
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Fixed Charge Derivatives
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Fixed Charge Derivatives
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Ok, so what good is it?
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ECD and Disulfide bonds
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ECD and Disulfide bonds
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Native ECD
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Unfolding probed by ECD yield
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Unfolding probed by ECD yield
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Unfolding probed by ECD yield
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Other labile modifications mapped by ECD

• Posphorylation
• N-glycosylation
• O-glycosylation
• Sialic acid residues on oligosaccharides
• Sulfation
• Hydrogen bonds noncovalent interactions

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Asparagine Deamidation
aspartic acid
isoaspartic acid or ß-aspartic acid
asparagine
The cannonical mechanism of deamidation involves
cyclization of asparagine (Asn) to form the
succinimide intermediate with loss of ammonium,
followed by hydration at either amide bond to
form a mixture of aspartic (Asp) and isoaspartic
(isoAsp) acids. Deamidation is irreversible
under physiological conditions, but the
isomerization of the products occurs at a
relatively slow rate.
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isoAspartyl cn58 fragment ion
cn58
zl-n-57
A B C
Aspartic Acid
Isoaspartic Acid
RAAAGADGDGAGADAR
Z Y
C6.58 m/z 572.3031
C8.58 m/z 744.3515
743.0
745.0
747.0

C13.58 m/z 1115.4956

1115.0
1117.0
1119.0
Mass/Charge (m/z)
Mass/Charge (m/z)
indicates secondary fragment ion due to loss of
NH3 and CHON from y14 (found in the spectra of
both peptides)
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Deamidated Tryptic Peptide of Cytochrome C
Asn31 of Cytochrome C is not exposed to the
solvent and cannot deamidate in the proteins
native state. The tryptic peptide
28TGPNLHGLFGR38 was found in the digest of Cyt.
C. The digest was incubated for 2 weeks at 37C
and pH 11 (20mM CAPS titrated w/ NH4OH) to fully
deamidate Asn31. The peptide (cleaned w/ POROS)
was then isolated and subjected to ECD.
Asn
Before incubation
3 Days
28TGPNLHGLFGR38, 2 ? D/isoD
7 Days
Asp
12 Days
cytochrome C
585
586
587
m/z
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ECD of deamidated Calmodulin tryptic peptide
c658
848.926 (1 ppm)
a
(M3H)2- Asp side chain
c
V F D K D G D G Y I S A A E L R

y
z
z10-57
m/z
c658
z10-57
60 Da loss ? D3, D5
1021.532 (1 ppm)
736.337 (1 ppm)
x7

c4
(M3H)3
c6
1020.5
1022.5
1024.5
736
737
738
m/z
m/z
z5-(leucine side chain)
(M2H)2
z6
c8
z142
z4
c5
z6
c7
c3
z10
z12
y6
z3
z8
w3
c10
y5
c11
c9
c12
z9
c13
c14
z11
y9
y3
z5
y7
c15
z13
a10
y8
y11
c458 D95 isoD95?
z12-57 D95 isoD95?
z14-57 D93 isoD93?
500
750
1000
1250
1500
1750
m/z
loss of NH3 and CO2 from (M3H)2. Note
internally calibrated on M3H3, M2H2, z12
and their isotopes.
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Abundance ratio of Asp/isoAsp diagnostic peaks
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Abundance ratios are linear
Plots of the zn-57 relative abundance versus
isoAsp content for ?, YWQHTADQFR-NH2 ?,
WAFDSAVAWR-NH2 , YDFIEYVR-NH2.
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ECD on an ion trap
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Electron-ion reaction methods

• Electron Capture Dissociation (ECD)
• Activated Ion ECD (AI-ECD)
• Hot ECD
• Electron Transfer Dissociation (ETD)
• Electron Detactment Dissociation (EDD)
• Electron Ionization Dissociation (EID or EIEIO)
• Electron Capture Induced Dissociation (ECID)

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Self Assessment

• How does ECD work? What fragments do you expect
  (mostly) from proteins and peptides?
• Why is it important to track the position of the
  radical?
• How can ECD be used to differentiate isomeric
  amino acids (2 examples)?
• Why do ECD rather than CAD?

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CH908 Mass spectrometry Lecture 1
Fini