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MS/MS Spectral Interpretation
Linda Breci
Chemistry Mass Spectrometry Facility
University of Arizona
MS Summer Workshop
MS/MS Spectral Interpretation
small molecule structure
Arpad Somogyi
Chemistry Mass Spectrometry Facility
University of Arizona
MS Summer Workshop
Session Overview
• Ways to approach predicting fragment ion formation
• Fragmentation examples
– Peptides
• Fragmentation mechanism
• Sequence a peptide
– Flavonoids
– Fatty Acids
– Oligonucleotides
MS/MS Fragmentation
Few libraries, little software available for data analysis
• Why?
We need useful information from MS/MS spectra
Few libraries, little software available for data analysis
• Why?
For MS/MS you have at least one of each of these:
•
Ionize
–
–
–
–
–
–
EI
CI
ESI
NSI
MALDI
FAB
•
Activate
–
–
–
–
–
–
CID
SID
SORI
IRMPD
ECD
BIRD
•
Analyze
–
–
–
–
–
–
–
Q
Q-trap
linear-trap
B sectors
E sectors
FTICR
TOF
You put them together like this:
* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSectorSID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF *
NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF *
EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q *
MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESIEBSector-CID-EBSector * and on…and on…
You put them together like this:
* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSectorSID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF *
NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF *
EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q *
MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESIEBSector-CID-EBSector * and on…and on…
And you buy them from different manufacturers
– Different source designs
• Example: ESI capillary temperature
– Different analyzer designs
• Example: Gas pressure, length of ion path (D timeframe)
How ions will fragment must be considered from
fundamentals (rather than rules)
• Ways to approach predicting MS/MS fragment formation
• Literature
– Study methods and ID’d spectra for your ion class
• Likely sites of protonation (or deprotonation)
– Find proton affinities or acid strengths
• Mobility of protons
– Consider the likelihood of multiple cleavage sites
– Consider multiple gas-phase configurations
• Likely leaving groups
Types of ions formed
• EI (hard ionization)
– M+· Radical ion
– A lot of fragmentation occurs upon ionization
• CI, FAB, ESI, APCI, MALDI (soft ionization)
– [M+H]+ Protonated ion
– [M-H]- Deprotonated ion
– [M+Na]+ and other metal cations
Today’s Topic
EI is not an MS/MS method
• Discussed Day 4
• Libraries of EI spectra are useful
• NIST/EPA/NIH Mass Spectral Library with Search
http://webbook.nist.gov/chemistry/
• Libraries are not always helpful, tutorials available
– http://www.chem.arizona.edu/massspec/
2 Categories of fragments from protonated or
deprotonated molecules
(CI, FAB, ESI, APCI, MALDI)
• Charge Remote
– Fragmentation reactions uninfluenced by charge
– High energy process
– Charge remote references provided
• Charge Directed
– Bond cleavage occurs with involvement of charge
– Low energy
– Most informative for many molecules
Today’s Topic
How ions will fragment must be considered from
fundamentals (rather than rules)
• Literature
– Study methods and ID’d spectra for your ion class
• Likely sites of protonation (or deprotonation)
– Find proton affinities or acid strengths
• Mobility of protons
– Consider the likelihood of multiple cleavage sites
– Consider multiple gas-phase configurations
• Likely leaving groups
How ions will fragment must be considered from
fundamentals (rather than rules)
• Literature
– Study methods and ID’d spectra for your ion class
• Likely sites of protonation (or deprotonation)
– Find proton affinities or acid strengths
• Mobility of protons
– Consider the likelihood of multiple cleavage sites
– Consider multiple gas-phase configurations
• Likely leaving groups
Fragmentation is a multi-step process
Step #1: Create Ions (add 1 or more protons)
ELECTROSPRAY
O
O
NH
H
H2N
NH
N
O
OH
NH
O
O
Fragmentation is a multi-step process
Step #1: Create Ions (add 1 or more protons)
ELECTROSPRAY
O
O
NH
H
H2N
NH
OH
N
O
NH
O
O
Step #2: Add energy (activation)
O
O
NH
H
H2N
NH
OH
N
SID
NH
O
O
H
O
O
O
NH
O
H
H2N
NH
N
O
CID
O
NH
OH
NH
O
O
H2N
NH
N
O
OH
NH
O
O
Fragmentation is a multi-step process
Step #3: Charge Directed Cleavage
H
O
O
NH
H2N
NH
OH
N
O
NH
O
Neutral + Fragment ion
O
b2
b3
a2
a3
What Rare
the likely
sites
of proton
location?
O
R3
O
R5
1
NH
NH
H2N
OH
N
O
R2
NH
O
y3
R4
y2
O
Model possible sites of proton location
(or loss of H) in Serine
O
H2N
CH
C
OH
CH2
OH
M + H → [M+H]+
DHrxn = -PA (M)
M → [M - H]- + H+
DHrxn = DHacid (M)
Model possible sites of proton location
(or loss of H) in Serine
O
Model with CH3NH2
(methyl amine)
H2N
CH
C
OH
CH2
OH
M + H → [M+H]+
DHrxn = -PA (M)
M → [M - H]- + H+
DHrxn = DHacid (M)
Model possible sites of proton location
(or loss of H) in Serine
O
Model with CH3NH2
(methyl amine)
H2N
CH
C
OH
CH2
methyl amine
PA
DH acid
214.9
402.0
OH
M + H → [M+H]+
DHrxn = -PA (M)
M → [M - H]- + H+
DHrxn = DHacid (M)
Ref: NIST
Model possible sites of proton location
(or loss of H) in Serine
Model with CH3COOH
(acetic acid)
O
Model with CH3NH2
(methyl amine)
H2N
CH
C
OH
CH2
OH
M + H → [M+H]+
DHrxn = -PA (M)
M → [M - H]- + H+
DHrxn = DHacid (M)
PA
DH acid
methyl amine
214.9
402.0
acetic acid
187.3
348.1
Ref: NIST
Model possible sites of proton location
(or loss of H) in Serine
Model with CH3COOH
(acetic acid)
O
Model with CH3NH2
(methyl amine)
H2N
CH
C
OH
CH2
Model with CH3OH
(methanol)
OH
M + H → [M+H]+
DHrxn = -PA (M)
M → [M - H]- + H+
DHrxn = DHacid (M)
PA
DH acid
methyl amine
214.9
402.0
acetic acid
187.3
348.1
methanol
180.3
382.0
Ref: NIST
Model possible sites of proton location
(or loss of H) in Serine
Model with CH3COOH
(acetic acid)
O
Model with CH3NH2
(methyl amine)
H2N
CH
C
OH
CH2
Model with CH3OH
(methanol)
M+H→
[M+H]+
M → [M - H]- + H+
OH
DHrxn = -PA (M)
DHrxn = DHacid (M)
PA
DH acid
methyl amine
214.9
402.0
acetic acid
187.3
348.1
methanol
180.3
382.0
Sites of Likely
protonation: NH2 > COOH > OH
deprotonation: COOH > OH > NH2
Ref: NIST
How ions will fragment must be considered from
fundamentals (rather than rules)
• Literature
– Study methods and ID’d spectra for your ion class
• Likely sites of protonation (or deprotonation)
– Find proton affinities or acid strengths
• Mobility of protons
– Consider the likelihood of multiple cleavage sites
– Consider multiple gas-phase configurations
• Likely leaving groups
Proton mobility
• Intramolecular proton transfer influences
– number of site-directed fragmentations
– amount of energy required for fragmentation
• Intramolecular proton transfer affected by
– site basicity
– gas-phase configuration
• Examples that follow:
– Spectra of increasingly basic peptides
– Overview chart demonstrating proton mobility (or lack of)
– Spectra of peptide conformers
50 eV
(SID)
Compare
Gas Phase Basicity
Arg (R):
240.6 kcal/mol
40 eV
(SID)
Lys (K):
227.3 kcal/mol
40 eV
(SID)
His (H):
227.3 kcal/mol
Ref: Gu, 1999
Pairwise bond cleavage between amino acids (Xxx-Zzz)
Zzz
10
Most
Abundant
9
8
7
6
5
4
3
2
1
Least
Abundant
Peptides with more basic Arg (R) vs. Lys (K)
.............R
1+
.............K
A D E F G H I L M N P Q S T V Y
A
D
E
F
G
H
I
L
M
N
P
Q
S
T
V
Y
A D E F G H I L M N P Q S T V Y
10
9
8
7
6
5
4
3
2
1
A
D
E
F
G
H
I
L
M
N
P
Q
S
T
V
Y
Prediction based on model peptides: Selective
Cleavage at Asp-Xxx will depend on number of
“Mobile” Protons
H
His
Asp
H
Arg (Lys)
or Arg or Lys
H
Asp
H
Arg (Lys)
Huang, Wysocki, Tabb, Yates Int. J. Mass Spectrom. 219, (1), 233-244, 2002
Peptides with basic Arg (R) 1 proton vs. 2 protons
1+
.............R
A D E F G H I L M N P Q S T V Y
A
D
E
F
G
H
I
L
M
N
P
Q
S
T
V
Y
2+
A D E F G H I L M N P Q S T V Y
10
9
8
7
6
5
4
3
2
1
A
D
E
F
G
H
I
L
M
N
P
Q
S
T
V
Y
Gas-phase conformation influences MS-MS
spectra observed
O
C
O
H2N
CH
CH3
C
O
H
N
CH
C
O
O
H
N
CH
CH3
C
H
N
CH
C
OH
CH3
N
CH3
Ala-Ala-Pro-Ala-Ala
Most Natural occurring amino acids have L configuration
at the chiral center (stereospecific biosynthesis)
Calculated structure of [AAPAA + H]+
Many sites of possible interaction
No solvent in the gas phase!
Gas-phase confirmation can influence MS-MS
spectra observed
Peptides containing proline stereoisomers fragment differently
All L-amino acids
except central residue
AVDPLG
All L-amino acids
1+
SID spectra of [AV(L)PLG+H] (29eV)
350
1+
SID spectra of [AV(D)PLG+H] (29eV)
y3
b4
100
300
80
y3
250
60
200
MH
150
+
40
100
+
MH
PL
50
PL
20
b4
0
a4
b3
0
0
100
200
300
m/z
400
500
0
100
200
300
m/z
400
500
Gas-phase confirmation can influence MS-MS
spectra observed
Peptides containing proline stereoisomers fragment differently
All L-amino acids
except central residue
AVDPLG
All L-amino acids
1+
SID spectra of [AV(L)PLG+H] (29eV)
350
1+
SID spectra of [AV(D)PLG+H] (29eV)
y3
b4
100
300
80
y3
250
60
200
MH
150
+
40
100
+
MH
PL
50
PL
20
b4
0
a4
b3
0
0
100
200
300
m/z
400
500
0
100
200
300
m/z
400
500
Gas-phase confirmation can influence MS-MS
spectra observed
Peptides containing proline stereoisomers fragment differently
All L-amino acids
except central residue
AVDPLG
All L-amino acids
1+
SID spectra of [AV(L)PLG+H] (29eV)
350
1+
SID spectra of [AV(D)PLG+H] (29eV)
y3
b4
100
300
80
y3
250
60
200
MH
150
+
40
100
+
MH
PL
50
PL
20
b4
0
a4
b3
0
0
100
200
300
m/z
400
500
0
100
200
300
m/z
400
500
Statistical analysis of cleavage at the Xxx-Pro bond
0.40
0.35
0.30
0.25
0.20
0.15
0.10
Ile
Le
u
Ly
s
Gl
u
Ph
e
Ty
r
Al
a
Gl
n
Th
r
As
n
Ar
g
Tr
p
Se
r
Gl
y
Pr
o
0.05
V
al
Hi
s
As
p
Relative Intensity [(a+b+y)Xxx-Pro / (a+b+y)all]
0.45
Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971
Statistical analysis of cleavage at the Xxx-Pro bond
Asp, His = Selective cleavage residues
Val, Ile, Leu = Bulky aliphatic side chains
0.40
0.35
0.30
0.25
0.20
0.15
0.10
Ile
Le
u
Ly
s
Gl
u
Ph
e
Ty
r
Al
a
Gl
n
Th
r
As
n
Ar
g
Tr
p
Se
r
Gl
y
Pr
o
0.05
V
al
Hi
s
As
p
Relative Intensity [(a+b+y)Xxx-Pro / (a+b+y)all]
0.45
Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971
How ions will fragment must be considered from
fundamentals (rather than rules)
• Literature
– Study methods and ID’d spectra for your ion class
• Likely sites of protonation (or deprotonation)
– Find proton affinities or acid strengths
• Mobility of protons
– Consider the likelihood of multiple cleavage sites
– Consider multiple gas-phase configurations
• Likely leaving groups
Likely Leaving Groups
• Bond cleavage is dependent on various factors
including:
– Leaving Groups
– Neighboring group participation reactions
– Intermediates (ion-neutral complex)
• For [M+H]+ ions the leaving group is a neutral
– lower methyl cation affinity is one measure of likelihood
– Compilations available in the literature
– Related to proton affinity
(kcal/mol)
Ref: Bartmess, 1989
Proton Affinity vs. Methyl Cation Affinity
Ref: Bartmess, 1989
Some fragmentation studies & basics
• Few examples from literature
– Cannot talk about all classes of compounds
– These examples suggest problem solving approaches
• Examples:
– Peptides
• Fragmentation mechanism
• Sequence a peptide
– Flavonoids
– Fatty Acids
– Oligonucleotides
Peptides
• Product ion spectra contain many types of fragment ions
–
–
–
–
charge directed
charge remote
internal fragments
immonium ions
• Important for sequencing
–
–
–
–
–
amino acid determined from D mass between peaks in spectrum
“y” ions series
“b” ions series
immonium ions (identify amino acids in the peptide)
“a” ions (confirm “b” ion after a loss of CO, 28 amu)
• Presented here:
– peptide fragment ions
– a mechanism for fragment ion formation
– a peptide to sequence
Peptide fragment ions
c2
Peptide bond fragment ions
b2
a2
O
H2N
CH
C
H
N
H
CH
O
O
H
N
C
CH
H
C
H
O
H
N
CH
C
OH
H
z2
R
y2
H
x2
CH
H2N
N
C
CH
O
R'
Internal immonium ion
R
H2N
CH
Amino acid immonium ion
Protonation occurs at amide oxygen or nitrogen
O
(Peptide)
CH
R1
C
R2
H
N
CH
O
C
O
H
N
H
CH
C
(Peptide)
R3
Ref: Yalcin, 1996
Protonation occurs at amide oxygen or nitrogen
O
(Peptide)
CH
R1
C
R2
H
N
CH
O
C
O
H
H
N
CH
C
(Peptide)
R3
Ref: Wysocki, 2000
A mechanism of peptide fragmentation
(1) D positive charge
(2) Nucleophilic attack
O
(Peptide)
CH
R1
C
R2
H
N
CH
O
C
O
H
H
N
CH
C
(Peptide)
R3
Ref: Wysocki, 2000
A mechanism of peptide fragmentation
(1) D positive charge
(2) Nucleophilic attack
O
(Peptide)
CH
R2
H
N
C
O
CH
C
R1
(Peptide)
O
H
N
O
C
R3
R2
CH
R1
CH
O
H
H
N
(Peptide)
H
N
CH
C
(Peptide)
OH
CH3
(3) cyclic intermediate
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
H
N
(Peptide)
CH
O
H
N
O
R1
(3) cyclic intermediate
R2
CH
C
(Peptide)
OH
CH3
H
N
(Peptide)
R2
CH
R1
O
H
N
O
CH
C
(Peptide)
OH
CH3
(4) Rearrangement
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
H
N
(Peptide)
R2
CH
R1
O
H2
N
O
CH
C
(Peptide)
O
CH3
H
N
(Peptide)
CH
R1
O
R2
+
H2N
CH
C
(Peptide)
O
b oxazolone ion
O
R3
neutral
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
H
N
(Peptide)
R2
CH
R1
O
H2
N
O
CH
C
(Peptide)
O
CH3
N
(Peptide)
CH
R1
O
R2
+
H
H2N
CH
C
(Peptide)
O
oxazolone neutral
(or other structure)
O
R3
y ion
Ref: Wysocki, 2000
Peptide fragment ions
c2
Peptide bond fragment ions
b2
a2
O
H2N
CH
C
H
N
H
CH
O
O
H
N
C
CH
H
C
H
O
H
N
CH
C
OH
H
z2
R
y2
H
x2
CH
H2N
N
C
CH
O
R'
Internal immonium ion
R
H2N
CH
Amino acid immonium ion
Peptide Sequencing
amino acid
71 u.
115 u.
Ala
O
C
Asp
O
H
N
CH
CH3
C
O
H
N
CH
C
CH2
C
OH
O
H
N
mass
Alanine
ALA
A
71.09
Arginine
ARG
R
156.19
Aspartic Acid
ASP
D
115.09
Asparagine
ASN
N
114.11
Cysteine
CYS
C
103.15
Glutamic Acid
GLU
E
129.12
Glutamine
GLN
Q
128.14
Glycine
GLY
G
57.05
Histidine
HIS
H
137.14
Isoleucine
ILE
I
113.16
Leucine
LEU
L
113.16
Lysine
LYS
K
128.17
Methionine
MET
M
131.19
Phenylalanine
PHE
F
147.18
Proline
PRO
P
97.12
Serine
SER
S
87.08
Threonine
THR
T
101.11
Tryptophan
TRP
W
186.12
Tyrosine
TYR
Y
163.18
Valine
VAL
V
99.14
LEARNING CHECK
Peptide Sequencing Exercise
Ion Current
over 60 min
MS/MS
MS
Peptide precursor ions observed by
MS
calculation of MH+
571.2 m/z measured
x2
1,142.4 [M+2H]
- 1.0
1,141.4 [M+H]
[M+ 2H]2+
m/z = 571.2
MH+
m/z = 1141.3
895.25
MS-MS of 571.2
Peptide Sequencing
amino acid
71 u.
115 u.
Ala
O
C
Asp
O
H
N
CH
CH3
C
O
H
N
CH
C
CH2
C
OH
O
H
N
mass
Alanine
ALA
A
71.09
Arginine
ARG
R
156.19
Aspartic Acid
ASP
D
115.09
Asparagine
ASN
N
114.11
Cysteine
CYS
C
103.15
Glutamic Acid
GLU
E
129.12
Glutamine
GLN
Q
128.14
Glycine
GLY
G
57.05
Histidine
HIS
H
137.14
Isoleucine
ILE
I
113.16
Leucine
LEU
L
113.16
Lysine
LYS
K
128.17
Methionine
MET
M
131.19
Phenylalanine
PHE
F
147.18
Proline
PRO
P
97.12
Serine
SER
S
87.08
Threonine
THR
T
101.11
Tryptophan
TRP
W
186.12
Tyrosine
TYR
Y
163.18
Valine
VAL
V
99.14
895.25
895.25
895.25
895.25
F
Phe
895.25
G
Gly
F
Phe
895.25
T
Thr
G
Gly
F
Phe
895.25
D
Asp
T
Thr
G
Gly
F
Phe
895.25
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
Build the peptide:
selected peptide = 1141.4
Estimate the number of amino acids
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
__ __ __ __ __ __ __ __ __ __
Possibly 10 amino acids
Consider a y-ion series
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
__ __ __ __ __ __ __ __ __ __
1141
1141.4 selected MH+
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
__ __ __ __ __ __ __ __ __ __
1141
1042
1141.4 selected MH+
1042.6 Largest fragment observed
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
__ __ __ __ __ __ __ __ __ __
1141
1042
y series ions
N
Asn
1141.4 selected MH+
1042.6 Largest fragment observed
98.8 difference
Is there an amino acid with that mass?
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
__
__ __ __ __ __ __ __ __ __
1141
1042
y series ions
N
Asn
99 = Valine
The missing amino acid
What is the next mass observed?
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
__
1141
__ __ __ __ __ __ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
F
__ __
1141
__ __ __ __ __ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
__ __
__
1141
__ __ __ __ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
__ __
__ T
__
1141
__ __ __ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
D
__ __
__ T
__ __
1141
__ __ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__
1141
__ __ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__
1141
__ __ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
__ __
895
1042
y series ions
N
Asn
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
y series ions
N
Asn
__ __
262
If this is a y-ion series:
262 = smallest ion in the series
what does it represent?
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
__ __
262
All amino acids in table are peptide bond to peptide bond
y series ions
71 u.
N
Asn
D
Asp
115 u.
M
D
Ala
Met
Asp
O
C
T
G
Asp
Thr Gly
O
H
N
CH
CH3
C
F
Phe
O
H
N
CH
C
CH2
C
OH
O
H
N
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
__ __
262
We’re missing one N-terminal hydrogen
y series ions
71 u.
N
Asn
D
Asp
115 u.
M
D
Ala
Met
Asp
O
C
T
G
Asp
Thr Gly
O
H
N
H
CH
CH3
C
F
Phe
O
H
N
CH
C
CH2
C
OH
O
H
N
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
__ __
262
We’re missing one C-terminal OH Group
y series ions
71 u.
N
Asn
D
Asp
115 u.
M
D
Ala
Met
Asp
O
C
T
G
Asp
Thr Gly
O
H
N
H
CH
CH3
C
H
N
CH
O
OH
C
H
N
CH2
C
OH
F
Phe
O
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
__ __
262
And the ionizing proton
Total = 19 amu
y series ions
71 u.
N
Asn
D
Asp
115 u.
M
D
Ala
Met
Asp
O
C
T
G
Asp
Thr Gly
O
H
N
H
CH
CH3
C
H+
H
N
CH
O
OH
C
H
N
CH2
C
OH
F
Phe
O
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
y series ions
N
Asn
__ __
262
262 = smallest identified fragment
- 19 = mass of H + OH + H
243 = mass of missing amino acids
What amino acids?
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
y series ions
N
Asn
__ __
Hint:
Tryptic!
262
262 = smallest identified fragment
- 19 = mass of H + OH + H
243 = mass of missing amino acids
What amino acids?
D
Asp
M
Met
D
Asp
T
Thr
G
Gly
F
Phe
895.25
V
FG
DM
__ __
__ T
__ __
__ D
__ N
__
1141
895
1042
y series ions
N
Asn
__ __
262
87 = Serine
156 = Arginine
243
19 = mass of H + OH + H
262
D
M
D
Asp
Met
Asp
115 = Aspartic Acid
128 = Lysine
243
19 = mass of H + OH + H
262
T
G
F
Thr Gly
Phe
895.25
V
FG
DM
SR
__ __
__ T
__ __
__ D
__ N
__ __
__
1141
895
1042
y series ions
N
Asn
262
87 = Serine
156 = Arginine
243
19 = mass of H + OH + H
262
D
M
D
Asp
Met
Asp
T
Thr
G
Gly
F
Phe
Some fragmentation studies & basics
• Few examples from literature
– Cannot talk about all classes of compounds
– These examples suggest problem solving approaches
• Examples:
– Peptides
• Fragmentation mechanism
• Sequence a peptide
– Flavonoids
– Fatty Acids
– Oligonucleotides
Flavonoids
• Common secondary plant metabolite
– Including flavonoid aglycones, O-glycosides, C-glycosides (arrows)
• Need reliable methodology for analysis
Ref: Cuyckens 2004
Flavonoids
• Group classification, chalcone aglycones, etc.
Ref: Cuyckens 2004
Flavonoids
• Group classification, chalcone aglycones, etc.
• Reported structures
300
250
400
19
350
450
Ref: Cuyckens 2004
Ion nomenclature for flavonoid glycosides
(apigenin 7-O-rutinoside illustrated)
nomenclature suggested by Ma, 1997 and Domon,1988
Ion nomenclature for flavonoid glycosides
(apigenin 7-O-rutinoside illustrated)
A and B ions (retro-Diels-Alder reactions) are most diagnostic:
- provide number and type of substituents in A & B ring
Low-energy CID (Fab-Magnetic sector-Quadrupole)
flavone
typical
1,3B+
0,4B+
0,4B+-H O
2
flavonol
typical
0,2A+
0,2A+-CO
1,4A++2H
1,3B+-2H
luteolin
kempferol
Ref: Ma, 1997
Low-energy CID (Fab-Magnetic sector-Quadrupole)
luteolin (flavone)
kempferol (flavonol)
Low-energy CID (Fab-Magnetic sector-Quadrupole)
luteolin (flavone)
kempferol (flavonol)
Some fragmentation studies & basics
• Few examples from literature
– Cannot talk about all classes of compounds
– These examples suggest problem solving approaches
• Examples:
– Peptides
• Fragmentation mechanism
• Sequence a peptide
– Flavonoids
– Fatty Acids
– Oligonucleotides
Fatty Acids
• Fragments formed by cleavage at alkyl bond can occur by charge
remote fragmentation (generally at higher energies)
– High Energy: Sector (KeV)
– Low Energy: QQQ, Qtrap, FTICR
– Intermediate Energy: Sector hybrids, TOF/TOF (collision gas, i.e. Xe)
• Homolytic bond-fragmentation mechanism (C--C → C- + -C radicals)
• 1,4-H2 elimination mechanism (Jensen, Tomer, Gross, 1985)
– X = O- or OLi2+
Ref: Jensen, 1985
Fatty Acids
• H-atom cleavage CRF mechanism (Claeys & Van den Heuvel, 1994)
– X = OLi2+ or OBuLi+
Ref: Claeys, 1994
Stearic acid (ESI-Sector-OATOF, 400eV collision, Xe)
Ref: Griffiths, 2003
Oleic acid (ESI-Sector-OATOF, 400eV collision, Xe)
Ref: Griffiths, 2003
docosahexaenoic acid ANSA derivative
(Sector, 400eV collision, Xe)
Gaps due to double bond
Ref: Griffiths, 2003
docosahexaenoic acid ANSA derivative
(QQQ, 30 eV collision, Ar)
Gaps due to double bond
Ref: Griffiths, 2003
Some fragmentation studies & basics
• Few examples from literature
– Cannot talk about all classes of compounds
– These examples suggest problem solving approaches
• Examples:
– Peptides
• Fragmentation mechanism
• Sequence a peptide
– Flavonoids
– Fatty Acids
– Oligonucleotides
Oligonucleotides
• McLuckey Nomenclature for multiply charged anions
– Gentle collisional activation = base loss
– Moderate conditions = consecutive fragmentations
Ref: McLuckey, 1993
Comparison of activation methods
CAD (CID) vs. IRMPD (Quadrupole Ion trap)
Parent-3
IRMPD:
Low mass observed
- PO3-1
-base anions
-Complete coverage
Ref: Keller, 2004
Comparison of activation methods
CAD (CID) vs. IRMPD (Quadrupole Ion trap)
Parent-3
CAD:
Loss of base
-provides little info
-leads to backbone
cleavages
Complete coverage
IRMPD:
Low mass observed
- PO3-1
-base anions
-Complete coverage
Ref: Keller, 2004
Comparison of activation methods
CAD (CID) vs. IRMPD (Quadrupole Ion trap)
Parent-3
CAD:
Loss of base
-provides little info
-leads to backbone
cleavages
Complete coverage
IRMPD:
Low mass observed
- PO3-1
-base anions
-Complete coverage
Ref: Keller, 2004
Steps for interpretation of oligonucleotide mass spectra for
determination of sequence
Ref: Ni, 1996
Steps for interpretation of oligonucleotide mass spectra for
determination of sequence
Ref: Ni, 1996
Comments on steps to interpretation
Ref: Ni, 1996
Suggested Reading List & References
General MS/MS
NIST Chemistry WebBook http://webbook.nist.gov/chemistry/
Rossi, D.T., Sinz, M.W., Mass Spectrometry in Drug Discovery, 2002, Marcel Dekker, Inc., New
York, NY.
Bartmess, J.E., Gas-Phase Equilibrium Affinity Scales and Chemical Ionization MassSpectrometry, Mass Spec. Reviews,1989, 8:297-343. (Affinity Tables)
McCloskey, J.A., Ed., Tandem Mass Spectrometry, Methods in Enzymology, 1990, Vol 193,
Academic Press, N.Y.
Peptides
Gu, C., Somogyi, A., Wysocki, V.H., Medzihradszky, K.F., Fragmentation of protonated
oligopeptides XLDVLQ (X=L, H, K or R) by surface induced dissociation: additional
evidence for the ‘mobile proton’ model., Analytica Chem. Acta, 1999, 397:247-256
Yalcin, T., Csizmadia, I.G., Peterson, M.R., Harrison, The Structure and Fragmentation of Bn (n
≥ 3) Ions in Peptide Spectra., A.G., J. Am. Soc. Mass Spectrom., 1996, 6, 1164-1174.
Wysocki, V.H., Tsaprailis, G., Smith, L., Breci, L., Mobile and localized protons: a framework for
understanding peptide dissociation, J. Mass Spectrom., 2000, 35, 1399-1406.
Flavonoids
Cuyckens, F., Claeys, M., Mass spectrometry in the structural analysis of flavonoids, J. Mass
Spectrom. 2004; 39: 1–15.
Ma, Y.L., Li, Q.M., Van den Heuvel, H., Claeys, M., Characterization of flavone and flavonol
aglycones by collision-induced dissociation tandem mass spectrometry, RCMS, 1997, 11:
1357.
Suggested Reading List & References (2)
Domon, B., Costello, C.E., A systematic nomenclature for carbohydrate fragmentations in FABMS/MS spectra of glycoconjugates. Glycoconj. J., 1988, 5:397.
Fatty Acids & Charge Remote
Griffiths, W., Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids, Mass
Spec. Reviews, 2003, 22, 81-152.
Jensen, N.J., Tomer, K.B., Gross, M.L., Gas phase ion decomposition occurring remote to a charge
site, J.Am.Chem.Soc., 1985, 107:1863-1868.
Claeys M., Van den Heuvel, H., Radical processes in remote charge fragmentations of lithium
cationized long-chain alkenyl and alkadienyl salicylic acids, Biol. Mass Spec., 1994, 23:20-26.
Gross, M.L., Charge-remote fragmentations – method, mechanism and applications, Int.J.Mass
Spec.Ion Process., 1992, 118: 137-165.
Wysocki, V.H., Ross, M.M., Charge-remote fragmentation of gas-phase ions – mechanistic and
energetic considerations in the dissociation of long-chain functionalized alkanes and alkenes,
Int.J.Mass Spec.Ion Process, 1991, 179-211.
Oligonucleotides
McLuckey, S.A., Habibi-Goudarzi, S., Decompositions of multiply Charged Oligonucleotide Anions,
J.Am.Chem.Soc., 1993, 115:12085-12095.
Keller, K.M., Brodbelt, J.S., Collisionally activated dissociation and infrared multiphoton dissociation
of oligonucleotides in a quadrupole ion trap, Anal.Chem., 2004, 326:200-210.
Ni, J.S., Pomerantz, S.C., Rozenski, J., Zhang, Y.H., McCloskey, J.A., Interpretation of
oligonucleotide mass spectra for determination of sequence using electrospray ionization and
tandem mass spectrometry, Anal.Chem., 1996, 68:1989-1999.