MSMS Spectral Interpretation Linda Breci Chemistry Mass Spectrometry Facility University of Arizona

1
MS/MS Spectral InterpretationLinda
BreciChemistry Mass Spectrometry
FacilityUniversity of Arizona MS Summer
Workshop
2
MS/MS Spectral Interpretationsmall molecule
structureArpad SomogyiChemistry Mass
Spectrometry FacilityUniversity of Arizona MS
Summer Workshop
3
Session Overview

• Ways to approach predicting fragment ion
  formation
• Fragmentation examples
• Peptides
• Fragmentation mechanism
• Sequence a peptide
• Flavonoids
• Fatty Acids
• Oligonucleotides

4
MS/MS Fragmentation

• Few libraries, little software available for data
  analysis
• Why?

5
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

6
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 onand on
7
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 onand 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)

8
How ions will fragment must be considered from
fundamentals (rather than rules)

• Ways to approach predicting MS/MS fragment
  formation
• Literature
• Study methods and IDd 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

9
Types of ions formed

• EI (hard ionization)
• M Radical ion
• A lot of fragmentation occurs upon ionization
• CI, FAB, ESI, APCI, MALDI (soft ionization)
• MH Protonated ion
• M-H- Deprotonated ion
• MNa and other metal cations

10
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/

11
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

12
How ions will fragment must be considered from
fundamentals (rather than rules)

• Literature
• Study methods and IDd 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

13
How ions will fragment must be considered from
fundamentals (rather than rules)

• Literature
• Study methods and IDd 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

14
Fragmentation is a multi-step process
Step 1 Create Ions (add 1 or more protons)
ELECTROSPRAY
15
Fragmentation is a multi-step process
Step 1 Create Ions (add 1 or more protons)
ELECTROSPRAY
Step 2 Add energy (activation)
SID
CID
16
Fragmentation is a multi-step process
Step 3 Charge Directed Cleavage
Neutral Fragment ion
What are the likely sites of proton location?
17
Model possible sites of proton location(or loss
of H) in Serine
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
18
Model possible sites of proton location(or loss
of H) in Serine
Model with CH3NH2 (methyl amine)
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
19
Model possible sites of proton location(or loss
of H) in Serine
Model with CH3NH2 (methyl amine)
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
Ref NIST
20
Model possible sites of proton location(or loss
of H) in Serine
Model with CH3COOH (acetic acid)
Model with CH3NH2 (methyl amine)
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
Ref NIST
21
Model possible sites of proton location(or loss
of H) in Serine
Model with CH3COOH (acetic acid)
Model with CH3NH2 (methyl amine)
Model with CH3OH (methanol)
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
Ref NIST
22
Model possible sites of proton location(or loss
of H) in Serine
Model with CH3COOH (acetic acid)
Model with CH3NH2 (methyl amine)
Model with CH3OH (methanol)
Sites of Likely protonation NH2 gt COOH gt
OH deprotonation COOH gt OH gt NH2
M H ? MH DHrxn -PA (M) M ? M
- H- H DHrxn DHacid (M)
Ref NIST
23
How ions will fragment must be considered from
fundamentals (rather than rules)

• Literature
• Study methods and IDd 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

24
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

25
Compare Gas 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)
Ref Gu, 1999
26
Pairwise bond cleavage between amino acids
(Xxx-Zzz)
27
Peptides with more basic Arg (R) vs. Lys (K)
.............R 1
.............K
28
Prediction based on model peptides Selective
Cleavage at Asp-Xxx will depend on number of
Mobile Protons
Huang, Wysocki, Tabb, Yates Int. J. Mass
Spectrom. 219, (1), 233-244, 2002
29
Peptides with basic Arg (R) 1 proton vs. 2
protons 1 .............R
2
30
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)
31
Calculated structure of AAPAA HMany sites
of possible interaction
No solvent in the gas phase!
32
Gas-phase confirmation can influence MS-MS
spectra observedPeptides containing proline
stereoisomers fragment differently
All L-amino acids except central residue AVDPLG
All L-amino acids
33
Gas-phase confirmation can influence MS-MS
spectra observedPeptides containing proline
stereoisomers fragment differently
All L-amino acids except central residue AVDPLG
All L-amino acids
34
Gas-phase confirmation can influence MS-MS
spectra observedPeptides containing proline
stereoisomers fragment differently
All L-amino acids except central residue AVDPLG
All L-amino acids
35
Statistical analysis of cleavage at the Xxx-Pro
bond
Breci, Tabb, Yates, Wysocki, (2003)
Analytical Chem. 751963-1971
36
Statistical analysis of cleavage at the Xxx-Pro
bond
Asp, His Selective cleavage residues Val, Ile,
Leu Bulky aliphatic side chains
Breci, Tabb, Yates, Wysocki, (2003)
Analytical Chem. 751963-1971
37
How ions will fragment must be considered from
fundamentals (rather than rules)

• Literature
• Study methods and IDd 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

38
Likely Leaving Groups

• Bond cleavage is dependent on various factors
  including
• Leaving Groups
• Neighboring group participation reactions
• Intermediates (ion-neutral complex)
• For MH 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

39
Ref Bartmess, 1989
(kcal/mol)
40
Proton Affinity vs. Methyl Cation Affinity
Ref Bartmess, 1989
41
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

42
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

43
Peptide fragment ions
c2
Peptide bond fragment ions
b2
a2
z2
y2
x2
Internal immonium ion
Amino acid immonium ion
44
Protonation occurs at amide oxygen or nitrogen
Ref Yalcin, 1996
45
Protonation occurs at amide oxygen or nitrogen
Ref Wysocki, 2000
46
A mechanism of peptide fragmentation
(1) D positive charge
(2) Nucleophilic attack
Ref Wysocki, 2000
47
A mechanism of peptide fragmentation
(1) D positive charge
(2) Nucleophilic attack
(3) cyclic intermediate
Ref Wysocki, 2000
48
A logical mechanism of peptide fragmentation
(3) cyclic intermediate
(4) Rearrangement
Ref Wysocki, 2000
49
A logical mechanism of peptide fragmentation

b oxazolone ion
neutral
Ref Wysocki, 2000
50
A logical mechanism of peptide fragmentation

oxazolone neutral (or other structure)
y ion
Ref Wysocki, 2000
51
Peptide fragment ions
c2
Peptide bond fragment ions
b2
a2
z2
y2
x2
Internal immonium ion
Amino acid immonium ion
52
Peptide Sequencing
71 u.
115 u.
Ala
Asp
53
LEARNING CHECKPeptide Sequencing Exercise
54
Ion Current over 60 min
MS/MS
MS
55
Peptide precursor ions observed by MS
calculation of MH 571.2 m/z measured x
2 1,142.4 M2H - 1.0 1,141.4 MH
M 2H2 m/z 571.2
MH m/z 1141.3
56
895.25
MS-MS of 571.2
57
Peptide Sequencing
71 u.
115 u.
Ala
Asp
58
895.25
59
895.25
60
895.25
61
895.25
F Phe
62
895.25
F Phe
G Gly
63
895.25
F Phe
G Gly
T Thr
64
895.25
F Phe
G Gly
T Thr
D Asp
65
895.25
F Phe
G Gly
T Thr
D Asp
M Met
66
895.25
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
67
895.25
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
68
895.25
Build the peptide selected peptide
1141.4 Estimate the number of amino acids
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
69
895.25

__ __ __ __ __ __ __ __ __ __
Possibly 10 amino acids Consider a y-ion series
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
70
895.25

__ __ __ __ __ __ __ __ __ __
1141
1141.4 selected MH
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
71
895.25

__ __ __ __ __ __ __ __ __ __
1141
1042
1141.4 selected MH 1042.6 Largest fragment
observed
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
72
895.25

__ __ __ __ __ __ __ __ __ __
1141
1042
1141.4 selected MH 1042.6 Largest fragment
observed 98.8 difference Is there an amino
acid with that mass?
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
73
895.25

V
__ __ __ __ __ __ __ __ __ __
1141
1042
99 Valine The missing amino acid What is the
next mass observed?
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
74
895.25

V
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
75
895.25

V F
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
76
895.25

V F G
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
77
895.25

V F G T
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
78
895.25

V F G T D
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
79
895.25

V F G T D M
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
80
895.25

V F G T D M D
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
81
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
82
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
If this is a y-ion series 262 smallest ion in
the series what does it represent?
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
83
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
All amino acids in table are peptide bond to
peptide bond
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
84
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
Were missing one N-terminal hydrogen
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
H
85
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
Were missing one C-terminal OH Group
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
OH
H
86
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
And the ionizing proton Total 19
amu
y series ions
H
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
OH
H
87
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
262 smallest identified fragment - 19 mass of
H OH H 243 mass of missing amino acids
What amino acids?
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
88
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
Hint Tryptic!
1141
895
1042
262
262 smallest identified fragment - 19 mass of
H OH H 243 mass of missing amino acids
What amino acids?
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
89
895.25

V F G T D M D N
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
87 Serine 156 Arginine 243 19 mass of
H OH H 262
115 Aspartic Acid 128 Lysine 243 19 mass
of H OH H 262
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
90
895.25

V F G T D M D N S R
__ __ __ __ __ __ __ __ __ __
1141
895
1042
262
87 Serine 156 Arginine 243 19 mass of
H OH H 262
y series ions
F Phe
G Gly
T Thr
D Asp
M Met
D Asp
N Asn
91
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

92
Flavonoids

• Common secondary plant metabolite
• Including flavonoid aglycones, O-glycosides,
  C-glycosides (arrows)
• Need reliable methodology for analysis

Ref Cuyckens 2004
93
Flavonoids

• Group classification, chalcone aglycones, etc.

Ref Cuyckens 2004
94
Flavonoids

• Group classification, chalcone aglycones, etc.
• Reported structures

300
250
400
19
350
450
Ref Cuyckens 2004
95
Ion nomenclature for flavonoid glycosides(apigeni
n 7-O-rutinoside illustrated)
nomenclature suggested by Ma, 1997 and Domon,1988
96
Ion nomenclature for flavonoid glycosides(apigeni
n 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
97
Low-energy CID (Fab-Magnetic sector-Quadrupole)
flavonol typical 0,2A 0,2A-CO 1,4A2H 1,3B-2H
flavone typical 1,3B 0,4B 0,4B-H2O
kempferol
luteolin
Ref Ma, 1997
98
Low-energy CID (Fab-Magnetic sector-Quadrupole)
luteolin (flavone)
kempferol (flavonol)
99
Low-energy CID (Fab-Magnetic sector-Quadrupole)
luteolin (flavone)
kempferol (flavonol)
100
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

101
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
102
Fatty Acids

• H-atom cleavage CRF mechanism (Claeys Van den
  Heuvel, 1994)
• X OLi2 or OBuLi

Ref Claeys, 1994
103
Stearic acid (ESI-Sector-OATOF, 400eV collision,
Xe)
Ref Griffiths, 2003
104
Oleic acid (ESI-Sector-OATOF, 400eV collision,
Xe)
Ref Griffiths, 2003
105
docosahexaenoic acid ANSA derivative (Sector,
400eV collision, Xe)
Gaps due to double bond
Ref Griffiths, 2003
106
docosahexaenoic acid ANSA derivative (QQQ, 30 eV
collision, Ar)
Gaps due to double bond
Ref Griffiths, 2003
107
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

108
Oligonucleotides

• McLuckey Nomenclature for multiply charged anions
• Gentle collisional activation base loss
• Moderate conditions consecutive fragmentations

Ref McLuckey, 1993
109
Comparison of activation methodsCAD (CID) vs.
IRMPD (Quadrupole Ion trap)
Parent-3

• IRMPD
• Low mass observed
• PO3-1
• base anions
• Complete coverage

Ref Keller, 2004
110
Comparison of activation methodsCAD (CID) vs.
IRMPD (Quadrupole Ion trap)

• CAD
• Loss of base
• provides little info
• leads to backbone
• cleavages
• Complete coverage

Parent-3

• IRMPD
• Low mass observed
• PO3-1
• base anions
• Complete coverage

Ref Keller, 2004
111
Comparison of activation methodsCAD (CID) vs.
IRMPD (Quadrupole Ion trap)

• CAD
• Loss of base
• provides little info
• leads to backbone
• cleavages
• Complete coverage

Parent-3

• IRMPD
• Low mass observed
• PO3-1
• base anions
• Complete coverage

Ref Keller, 2004
112
Steps for interpretation of oligonucleotide mass
spectra for determination of sequence
Ref Ni, 1996
113
Steps for interpretation of oligonucleotide mass
spectra for determination of sequence
Ref Ni, 1996
114
Comments on steps to interpretation
Ref Ni, 1996
115
Suggested Reading List References

• General MS/MS
• NIST Chemistry WebBook http//webbook.nist.gov/c
  hemistry/
• 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, 8297-343. (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 (XL, H, K or R) by surface
  induced dissociation additional evidence for the
  mobile proton model., Analytica Chem. Acta,
  1999, 397247-256
• 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 39 115.
• 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.

116
Suggested Reading List References (2)

• Domon, B., Costello, C.E., A systematic
  nomenclature for carbohydrate fragmentations in
  FAB-MS/MS spectra of glycoconjugates. Glycoconj.
  J., 1988, 5397.
• 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, 1071863-1868.
• 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,
  2320-26.
• Gross, M.L., Charge-remote fragmentations
  method, mechanism and applications, Int.J.Mass
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