ms/ms spectral interpretation linda breci chemistry mass spectrometry facility university of arizona...
Post on 21-Dec-2015
222 views
TRANSCRIPT
MS/MS Spectral Interpretation
Linda BreciChemistry Mass Spectrometry Facility
University of Arizona
MS Summer Workshop
MS/MS Spectral Interpretationsmall molecule structure
Arpad SomogyiChemistry 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
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:
• Analyze– Q– Q-trap– linear-trap– B sectors– E sectors– FTICR– TOF
• Activate– CID– SID– SORI– IRMPD– ECD– BIRD
• Ionize– EI– CI– ESI– NSI– MALDI– FAB
You put them together like this:
* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-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 * ESI-
EBSector-CID-EBSector * and on…and on…
You put them together like this:
* ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-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 * ESI-
EBSector-CID-EBSector * and on…and on…
– Different source designs• Example: ESI capillary temperature
– Different analyzer designs• Example: Gas pressure, length of ion path ( timeframe)
And you buy them from different manufacturers
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
H2NNH
NNH
NHOH
O
O
O
O
O
H
Step #1: Create Ions (add 1 or more protons)
ELECTROSPRAY
Fragmentation is a multi-step process
H2NNH
NNH
NHOH
O
O
O
O
O
H
H2NNH
NNH
NHOH
O
O
O
O
O
H
H2NNH
NNH
NHOH
O
O
O
O
O
H
H2NNH
NNH
NHOH
O
O
O
O
O
H
Step #1: Create Ions (add 1 or more protons)
Step #2: Add energy (activation)
SID
CID
ELECTROSPRAY
H2NNH
NNH
NHOH
O
O
O
O
O
H
y3
b2
a2
H2NNH
NNH
NHOH
O
O
O
O
O
R1
R2
R3
R4
R5
y2
b3
a3
Step #3: Charge Directed Cleavage
Fragmentation is a multi-step process
Neutral + Fragment ion
What are the likely sites of proton location?
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
Model with CH3NH2
(methyl amine)
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
Model with CH3NH2
(methyl amine)
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Ref: NIST
PA H acid 402.0214.9methyl amine
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
Model with CH3COOH(acetic acid)
Model with CH3NH2
(methyl amine)
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Ref: NIST
PA H acid
348.1187.3acetic acid
402.0214.9methyl amine
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
Model with CH3COOH(acetic acid)
Model with CH3NH2
(methyl amine)
Model with CH3OH(methanol)
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Ref: NIST
PA H acid
382.0180.3methanol
348.1187.3acetic acid
402.0214.9methyl amine
Model possible sites of proton location(or loss of H) in Serine
H2N CH C
CH2
OH
O
OH
Model with CH3COOH(acetic acid)
Model with CH3NH2
(methyl amine)
Model with CH3OH(methanol)
M + H → [M+H]+ Hrxn = -PA (M)
M → [M - H]- + H+ Hrxn = Hacid (M)
Ref: NIST
PA H acid
382.0180.3methanol
348.1187.3acetic acid
402.0214.9methyl amine
Sites of Likelyprotonation: NH2 > COOH > OHdeprotonation: COOH > OH > NH2
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
Ref: Gu, 1999
CompareGas Phase Basicity
Arg (R):240.6 kcal/mol
Lys (K):227.3 kcal/mol
His (H):227.3 kcal/mol
50 eV(SID)
40 eV(SID)
40 eV(SID)
Pairwise bond cleavage between amino acids (Xxx-Zzz)
Z z z
1
2
3
4
5
6
7
8
9
10 Most Abundant
LeastAbundant
Prediction based on model peptides: Selective Cleavage at Asp-Xxx will depend on number of
“Mobile” Protons
H H
or Arg or Lys
His Arg (Lys)Asp
H HArg (Lys)
Asp
H HArg (Lys)
Asp
Huang, Wysocki, Tabb, Yates Int. J. Mass Spectrom. 219, (1), 233-244, 2002
H2N CH C
CH3
O
HN CH C
CH3
O
N
C
O
HN CH C
CH3
O
HN CH C
CH3
OH
O
Gas-phase conformation influences MS-MS spectra observed
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 acidsAll L-amino acids
except central residueAVDPLG
0 100 200 300 400 5000
20
40
60
80
100
MH+
PLb
3
y3
a4
b4
SID spectra of [AV(D)
PLG+H]1+ (29eV)
m/z
0 100 200 300 400 5000
50
100
150
200
250
300
350
PL
y3
MH+
b4
SID spectra of [AV(L)
PLG+H]1+ (29eV)
m/z
Gas-phase confirmation can influence MS-MS spectra observed
Peptides containing proline stereoisomers fragment differently
All L-amino acidsAll L-amino acids
except central residueAVDPLG
0 100 200 300 400 5000
20
40
60
80
100
MH+
PLb
3
y3
a4
b4
SID spectra of [AV(D)
PLG+H]1+ (29eV)
m/z
0 100 200 300 400 5000
50
100
150
200
250
300
350
PL
y3
MH+
b4
SID spectra of [AV(L)
PLG+H]1+ (29eV)
m/z
Gas-phase confirmation can influence MS-MS spectra observed
Peptides containing proline stereoisomers fragment differently
All L-amino acidsAll L-amino acids
except central residueAVDPLG
0 100 200 300 400 5000
20
40
60
80
100
MH+
PLb
3
y3
a4
b4
SID spectra of [AV(D)
PLG+H]1+ (29eV)
m/z
0 100 200 300 400 5000
50
100
150
200
250
300
350
PL
y3
MH+
b4
SID spectra of [AV(L)
PLG+H]1+ (29eV)
m/z
Statistical analysis of cleavage at the Xxx-Pro bond
Val HisAsp Ile Leu Lys Glu Phe Tyr Ala Gln Thr Asn Arg Trp Ser Gly Pro
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Rel
ativ
e In
tens
ity [
(a+
b+y)
Xxx
-Pro /
(a+
b+y)
all]
Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971
Statistical analysis of cleavage at the Xxx-Pro bond
Val HisAsp Ile Leu Lys Glu Phe Tyr Ala Gln Thr Asn Arg Trp Ser Gly Pro
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Rel
ativ
e In
tens
ity [
(a+
b+y)
Xxx
-Pro /
(a+
b+y)
all]
Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971
Asp, His = Selective cleavage residuesVal, Ile, Leu = Bulky aliphatic side chains
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
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 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 bond fragment ions
Peptide fragment ions
H2N CH C
H
O
HN CH C
H
O
HN CH C
H
O
HN CH C
H
OH
O
CH
R
H2N C
N
CH
R'O
H
Internal immonium ion Amino acid immonium ion
a2
b2
c2
x2
y2
z2
H2N CH
R
Protonation occurs at amide oxygen or nitrogen
Ref: Yalcin, 1996
(Peptide) CH C
R1
O
N CH C N CH C
R3
(Peptide)
O
O
R2
H H
H
Protonation occurs at amide oxygen or nitrogen
Ref: Wysocki, 2000
(Peptide) CH C
R1
O
N CH C N CH C
R3
(Peptide)
O
O
R2
H H
H
A mechanism of peptide fragmentation
(Peptide) CH C
R1
O
N CH C N CH C
R3
(Peptide)
O
O
R2
H H
H
(1) positive charge(2) Nucleophilic attack
Ref: Wysocki, 2000
A mechanism of peptide fragmentation
(3) cyclic intermediate
(Peptide) CH C
R1
O
N CH C N CH C
R3
(Peptide)
O
O
R2
H H
H
(1) positive charge(2) Nucleophilic attack
(Peptide) CH
R1O
N R2
OH
H
HN CH C
CH3
(Peptide)
O
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
(4) Rearrangement
(Peptide) CH
R1O
N R2
OH
H
HN CH C
CH3
(Peptide)
O
(Peptide) CH
R1O
HN R2
OH
HN CH C
CH3
(Peptide)
O
(3) cyclic intermediate
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
(Peptide) CH
R1O
HN R2
O
H2N CH C
CH3
(Peptide)
O
H2N CH C
R3
(Peptide)
O
(Peptide) CH
R1O
N R2
O
H
b oxazolone ion neutral
+
Ref: Wysocki, 2000
A logical mechanism of peptide fragmentation
oxazolone neutral(or other structure)
y ion
+
(Peptide) CH
R1O
HN R2
O
H2N CH C
CH3
(Peptide)
O
(Peptide) CH
R1O
N R2
O
H2N CH C
R3
(Peptide)
O
H
Ref: Wysocki, 2000
Peptide bond fragment ions
Peptide fragment ions
H2N CH C
H
O
HN CH C
H
O
HN CH C
H
O
HN CH C
H
OH
O
CH
R
H2N C
N
CH
R'O
H
Internal immonium ion Amino acid immonium ion
a2
b2
c2
x2
y2
z2
H2N CH
R
Peptide Sequencing
mass amino acid
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
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
Peptide precursor ions observed by MS
MH+
m/z = 1141.3
[M+ 2H]2+
m/z = 571.2
calculation of MH+
571.2 m/z measured x 2 1,142.4 [M+2H] - 1.0 1,141.4 [M+H]
Peptide Sequencing
mass amino acid
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
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
Build the peptide:selected peptide = 1141.4Estimate the number of amino acids
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
Possibly 10 amino acidsConsider a y-ion series
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
1141.4 selected MH+
y series ions
1141
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
1141.4 selected MH+
1042.6 Largest fragment observedy series ions
11411042
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
1141.4 selected MH+
1042.6 Largest fragment observed 98.8 differenceIs there an amino acid with that mass?
y series ions
11411042
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
99 = ValineThe missing amino acidWhat is the next mass observed?y series ions
11411042
V
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
895
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
If this is a y-ion series:262 = smallest ion in the serieswhat does it represent?
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
All amino acids in table are peptide bond to peptide bond
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
We’re missing one N-terminal hydrogen
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
H
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
We’re missing one C-terminal OH Group
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
OH
H
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
And the ionizing proton Total = 19 amu
C
O
HN CH C
CH3
O
HN CH C
CH2
O
C
OH
O
HN
71 u. 115 u.
Ala Asp
OH
H
H+
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
262 = smallest identified fragment- 19 = mass of H + OH + H243 = mass of missing amino acids What amino acids?
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
262 = smallest identified fragment- 19 = mass of H + OH + H243 = mass of missing amino acids What amino acids?
Hint:Tryptic!
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N
262895
87 = Serine156 = Arginine243 19 = mass of H + OH + H262
115 = Aspartic Acid128 = Lysine243 19 = mass of H + OH + H262
FPhe
GGly
TThr
DAsp
MMet
DAsp
NAsn
895.25
__ __ __ __ __ __ __ __ __ __
y series ions
11411042
V F G T D M D N S R
262895
87 = Serine156 = Arginine243 19 = mass of H + OH + H262
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. • Reported structures
400
450
350
300
19
250
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)
luteolin kempferol
flavonetypical1,3B+
0,4B+
0,4B+-H2O
flavonoltypical0,2A+
0,2A+-CO1,4A++2H1,3B+-2H
Ref: Ma, 1997
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
docosahexaenoic acid ANSA derivative (Sector, 400eV collision, Xe)
Ref: Griffiths, 2003
Gaps due to double bond
docosahexaenoic acid ANSA derivative (QQQ, 30 eV collision, Ar)
Ref: Griffiths, 2003
Gaps due to double bond
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 methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)
Ref: Keller, 2004
IRMPD:Low mass observed- PO3
-1
-base anions-Complete coverage
Parent-3
Comparison of activation methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)
Ref: Keller, 2004
CAD:Loss of base-provides little info -leads to backbone cleavagesComplete coverage
IRMPD:Low mass observed- PO3
-1
-base anions-Complete coverage
Parent-3
Comparison of activation methodsCAD (CID) vs. IRMPD (Quadrupole Ion trap)
Ref: Keller, 2004
CAD:Loss of base-provides little info -leads to backbone cleavagesComplete coverage
IRMPD:Low mass observed- PO3
-1
-base anions-Complete coverage
Parent-3
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
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 Mass-Spectrometry, 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
Domon, B., Costello, C.E., A systematic nomenclature for carbohydrate fragmentations in FAB-MS/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.
Suggested Reading List & References (2)