CH 908: Mass Spectrometry
Lecture 9
Electron Capture Dissociation of
Peptides and Proteins
Prof. Peter B. O’Connor
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
•
•
•
•
•
Utility
–
–
–
–
–
•
cyclic peptide data
radical migration
double resonance
radical traps
Disulphide bonds
Protein structural studies
Labile modifications
Isomer determination
HDX?
Other ExD methods
Odd vs. Even Electron
Fragmentation
• Even electron = proton rearrangements
• Odd electron = radical rearrangements
• Non-ergodic fragmentation = FAST!!
ECD Spectrum
Hot H• Mechanism of ECD
UW Mechanism
Activated-Ion ECD
Activated-Ion ECD
Activated-Ion ECD
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
“Hot” ECD
The effect of metals
In this case, Zinc binding killed
the electron – thus killing
fragmentation
Electron capture dissociation
of polyethylene glycol
HO-(CH2-CH2-O)n-H
Cyclic peptide structures
N
O
N
O
N
O
O
N
O
N
N
N
N
N
N
O
N
N
O
O
O
O
N
O
N
HO
O
O
N
N
O
N
N
N
O
N
O
O
N
N
N
O
N
N
N
N
O
N
N
O
O
N
O
N
N
O
N
N
O
O
•Two histidines
•Tryptophan
•phenylalanine
N
O
N
O
cyclo-LLFHWAVGH
N
O
O
O
gramicidin S
cyclosporin A
•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.
ECD of cyclo- LLFHWAVGH
X12
X30
(M+2H)2+

†

†
†
†
†
cleavage assignable to
backbone cleavage
cleavage assignable to loss
of H•, H2O, NH3, CO,
CONH
cleavage assignable to
sidechain fragmentation
†
†
†
††
†
††
†
†
†
†


†
†

†
†
†

†
†
†
††
†
300
400
 : electronic noise,  Harmonic
500
†
600
†
†
†
†
†
†
m/z
†
††
†
††
†
†
†
†
†
†† †
700
†
†
†
†
†
†
†
†

†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
††
† †
†
†
††
†
†
†
†
†
††
†
†
†
†
†
††
†
††
† †
† ††
†
†
†
†
†
800
900
†
†
††
†
†
†
†
†† †
†
1000
The free radical reaction cascade will continue until one of the following
occurs:
1.
The various energy losses in the reactions and in black body radiation
cool the system sufficiently that further rearrangments are impossible
2.
The free radical is eliminated leaving the charge containing peptide in
an even-electron state
3.
The free radical is stabilized at a site of low reactivity
Is the Free Radical Cascade
mechanism really correct?
1. 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
2. FRC mechanism implies that the radical intermediates are long lived (≈
microseconds or more). This can be tested by double resonance.
3. FRC mechanism implies radical migration. Addition of a radical trap
moiety to the peptide should radically change the fragmentation.
Free Radical Cascade in Linear Peptide ECD:
Deuterium scrambling by Hydrogen Abstraction
O
O
O
O
R1
C
CH
R1
NH
C
CD
R1
C
CD
NH
H
NH
CD
H
D
NH
CD
C
O
CH
C
NH
R2
CD
O
C
O
H
CH
C
NH
CD
R2
C
O
CH
C
R2
C
+
R1
C
CD
NH
NH
O
CH
C
R2
O
O
O
secondary
D mass = -1.006 Da
Deuterium substitution Isotopic scrambling
at sites where the
prior to secondary
radical migrates to
cleavage
primary
D
CD
C
O
ECD of D-labeled synthetic peptides:
H/D Scrambling
BUSM 1 = undeuterated
RAG2DADG2DDADG2DDAG2DAAR
c15
c12
1245
1240
BUSM 2 = deuterated
c9
800
990
985
c6
805
510
515
1250
1255
995
1000
805
810
515
520
1250
1255
995
1000
805
810
515
520
c14
c11
1175
1170
c8
925
930
935
c5
685
440
690
445
1180
1185
935
940
690
695
440
445
1180
1185
935
940
690
695
440
445
c13
c10
1055
1060
855
c7
860
Top: ECD of BUSM 1
625
630
Middle: 0.2 eV ECD of BUSM 2
1065
1070
860
865
630
635
Bottom: 9 eV ECD of BUSM 2
1065
1070
860
865
630
635
Spectra of BUSM 2 were shifted
left by 2 Daltons per glycine to
align with those of BUSM 1
H/D Scrambling Results
• The initial radical does migrate to αcarbons
• The best mechanism supports a hydrogen
bonded dimer as the long-lived radical
intermediate
Is the Free Radical Cascade
mechanism really correct?
1. 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
2. FRC mechanism implies that the radical intermediates are long lived (≈
microseconds or more). This can be tested by double resonance.
3. FRC mechanism implies radical migration. Addition of a radical trap
moiety to the peptide should radically change the fragmentation.
Double Resonance
+ = short lived
+
X
+
X
+
= long lived
+ = short lived
Resonantly Eject
Timeframe = 0.01 – 1 msec
Double Resonance
Which fragments come from long lived intermediates?
quench
inject
ions
isolate
ECD
Excite
Detect
Resonant
Excite
Fragments derived from long-lived intermediates will
disappear from spectra
Fragments from short-lived intermediates will not change
Substance P ECD
RPKPQQFFGLM-NH2
M2+
c5
w2
c4
*
c10
c7
c6
c8
a7
300
400
500
600
700
800
300
400
500
600
700
800
m/z
m/z
z9
[M+2H]+•
c9
900
1000
1100
1200
1300
1400
900
1000
1100
1200
1300
1400
*: electronic noise
Substance P
S
HN
O
N H2
N H2
N
H
H
NH
O
O
N H2
HN
O
O
O
N H2
N
N
H
N
O
c4
z9
O
O
NH
H
N
H
N
N
H
N
H
O
O
N H3+
O
O
N H2
BUSM 1 ECD
RAAA GADG DGAG ADAR
c6
c8
c5
c3
a4
w2
c4
a7
a6
a5
z7
z6
300
400
500
b7 y7
c7
a7
600
m/z
700
-34
-CO
-45
c15
-60
c12
c10
y9
z8
z9
b9 c
9
a9
a11
c11
a12
a13
c13
800
900
c14
z14
z12
z13
a10
1000
m/z
-104/5
-101
-149
-72
-78
1100
b14
a14
a15
z15
1200
-NH3
[M+2H]+•
-130
1300
1400
Is the Free Radical Cascade
mechanism really correct?
1. 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
2. FRC mechanism implies that the radical intermediates are long lived (≈
microseconds or more). This can be tested by double resonance.
3. FRC mechanism implies radical migration. Addition of a radical trap
moiety to the peptide should radically change the fragmentation.
Radical traps
Radical traps
Fixed Charge Derivatives
Fixed Charge Derivatives
Ok, so what good is it?
ECD and
Disulfide
bonds
ECD and
Disulfide
bonds
Native ECD
Unfolding probed by ECD yield
Unfolding
probed by
ECD yield
Unfolding
probed by
ECD yield
Other labile modifications mapped
by ECD
•
•
•
•
•
Posphorylation
N-glycosylation
O-glycosylation
Sialic acid residues on oligosaccharides
Sulfation
• Hydrogen bonds – noncovalent
interactions
Asparagine Deamidation
aspartic acid
asparagine
isoaspartic acid
or
β-aspartic acid
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.
isoAspartyl cn•+58 fragment ion
HN
e-
NH 3
HN
HN
H2N
HN
NH
O
OH
CH
N
H
H2N
O
O
NH
HN
COOH
N
H
H2N
O
O
Aspartic Acid
OH
OH
+
O
C
H2
NH
H2N
HN
H
O
HN
NH 3
CH
H2C
NH
cn•+58
A
B
C
HN
zl-n-57
Isoaspartic Acid
RAAAGADGDGAGADAR
Z
Y
C6.+58
m/z = 572.3031
572.0
574.0
576.0
572.0
574.0
576.0
C8.+58
m/z = 744.3515
743.0
745.0
747.0
743.0
745.0
747.0
#
C13.+58
m/z =1115.4956
1115.0
1117.0
Mass/Charge (m/z)
1119.0
#
1115.0
1117.0
1119.0
Mass/Charge (m/z)
# indicates secondary fragment ion due to loss of NH3 and CHON from y14 (found in the spectra of both peptides)
COOH
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 37°C 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,

D/isoD
2+
7 Days
Asp
12 Days
cytochrome C
585
586
m/z
587
ECD of deamidated Calmodulin tryptic peptide
848.926 (~1 ppm)
(M+3H)2+•- Asp side chain
c6•+58
a
c
VFDKDGDGY I SAAELR
†
y
848.5
849.5
m/z
60 Da loss 
z
850.5
D3,
z10-57
c6•+58
736.337 (~1 ppm)
D5
z10-57
1021.532 (~1 ppm)
x7
~(M+3H)
c4
3+
c6
736
z5•-(leucine side chain)
z6•
z4•
c5
c3
w3 z3•
y5
y3
y6
z5•
500
z14•2+
z6•
c7
750
D95 isoD95?
1020.5
738
1022.5
m/z
1024.5
(M+2H)2+
c8
z8•
y7
c4•+58
737
m/z
y8
m/z
z9•
y9
c9
1000
z10•
z12•
c10
c11
c12
z11•
a10 y11
z12-57
1250
D95 isoD95?
c13
z13•
z14-57
D93
isoD93?
c14
c15
1500
† loss of NH3 and CO2 from (M+3H)2+•. Note: internally calibrated on [M+3H]3+, [M+2H]2+, z12 and their isotopes.
1750
Abundance ratio of Asp/isoAsp
diagnostic peaks
Abundance ratios are linear
One calibration point
is always available.
Plots of the zn-57 relative abundance versus % isoAsp content for; ▲,
YWQHTADQFR-NH2 ; ♦, WAFDSAVAWR-NH2 ; ■, YDFIEYVR-NH2.
“ECD” on an ion trap
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)
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?
CH908: Mass spectrometry
Lecture 1
Fini…