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MS/MS Scan Modes Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS Summer Workshop

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Title: MS/MS Scan Modes Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS Summer Workshop


1
MS/MS Scan ModesLinda BreciChemistry Mass
Spectrometry FacilityUniversity of Arizona MS
Summer Workshop
2
Why are there different MS/MS scan modes
  • Increase selectivity (find analyte in complex
    mix)
  • Increase sensitivity (find low abundance analyte)
  • While keeping signal intensity as high as
    possible
  • Best Instrument type in general
  • No best type many successful MS/MS
    configurations
  • Best instrument type for specific scan modes
  • precursor ion, neutral loss, selected reaction
    monitoring scans
  • continuous source analyzer
  • Triple Quad (QQQ)

3
Analyzers
Quadrupole
TOF
Continuous source
Pulsed source
Quadrupole Ion trap
Source ions cut-off and then scanned
FTICR
Magnetic sector
Source ions cut-off and then scanned
Continuous source
4
Quadrupole (Q)
http//www-methods.ch.cam.ac.uk/meth/ms/theory/qua
drupole.html
5
Time-of-Flight (TOF)
http//www.kore.co.uk/MS-200_principles.htm
6
TOF
D
Detector
m/z
V
KE eV ½mv2                    m mass
                                                 
  V velocity       v
D/t                        D
distance of flight                               
             t  time of flight
½m(D/t)2 eV      KE
kinetic energy                                 
            e charge      
7
Quadrupole Ion Trap (QIT)
http//www-methods.ch.cam.ac.uk/meth/ms/theory/ion
trap.html
8
Fourier Transform-Ion Cyclotron Resonance (FTICR)
http//www-methods.ch.cam.ac.uk/meth/ms/theory/ion
trap.html
9
Magnetic forces move ions in a circular path
Analyzers based on magnetic fields Sector
demo http//www.casetechnology.com/implanter/m
agnet.html
10
Quadrupole (Q)
http//www-methods.ch.cam.ac.uk/meth/ms/theory/qua
drupole.html
11
Tandem in Space vs. Tandem in Time MS/MS
  • Space (Analyzers cannot trap, must be linked)
  • Triple Quad
  • TOF
  • Sector
  • Many combinations of linked analyzers possible
  • Time (Trapping analyzers)
  • Quadrupole Ion trap
  • ICR
  • Most activation by CID (CAD)

12
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

13
Single Scan in a Quadrupole (Q)
  • One rf/dc combination one m/z value to detector
  • remaining m/z values neutralized at the quads
  • sample loss is a disadvantage of scanning
    instruments
  • Scanning instrument ramps rf/dc voltage
    combinations

14
Quadrupole (Q) Single analyzer scan
Source
Detector
Q1
15
Quadrupole (Q) Single analyzer scan
Source
Detector
Q1
(1st Vac/Vdc)
16
Quadrupole (Q) Single analyzer scan
Source
Detector
Q1
(2nd Vac/Vdc)
17
Quadrupole (Q) Single analyzer scan
Source
Detector
Q1
(3rd Vac/Vdc)
18
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

19
Product Ion Scan
  • Qualitative structural information
  • aka Daughter ion scan
  • Data dependent product ion scan
  • Intense ion above selected threshold value
    selected in Q1
  • Q1 is used to select one m/z
  • This parent ion is dissociated in Q2 (Rf only)
  • Q2 in Rf only mode is high transmission device
  • Fragments (product ions) are formed by collisions
  • Product ions are scanned through Q3

20
Tandem in Space (QQQ) Product Ion Scan
Select one m/z
(fixed Vac/Vdc)
21
Tandem in Space (QQQ) Product Ion Scan
Dissociate
(collide with gas)
22
Tandem in Space (QQQ) Product Ion Scan
Scan Products
(scan Vac/Vdc)
23
Tandem in Time (Ion Trap) -- Product Ion Scan
24
Tandem in Time (Ion Trap) -- Product Ion Scan
Select one m/z
25
Tandem in Time (Ion Trap) -- Product Ion Scan
Dissociate
(collide with gas)
26
Tandem in Time (Ion Trap) -- Product Ion Scan
Scan Products
27
MS of a Peptide ( ) in an Ion trap
  • Scan Voltage Ramped
  • Sequential m/z hit detector

28
MS of a Peptide (YGGFL )
556.2
29
MS of a Peptide (YGGFL)
Select for MS-MS (YGGFL Parent)
556.2
30
MS-MS of a Peptide ( ) in an Ion trap
Scan Products
Dissociate
Select one m/z
31
MS-MS of a Peptide (YGGFL, m/z 556.2)
425
556.2
538
397
279
336
32
MS-MS of a Peptide (YGGFL, m/z 556.2)
b4
b4
425
y2
556.2
y3
-H2O
a4
538
397
y3
y2
279
336
33
MS-MS of a Peptide (YGGFL, m/z 556.2)
b4
b4
425
y2
556.2
y3
Select for MS-MS-MS (YGGFL Fragment)
-H2O
a4
538
397
y3
y2
279
336
34
MS-MS-MS (MSn) of a Fragment ( ) in an Ion
trap
Source
Time 1
Time 3
Time 2
Select one m/z
Dissociate
Detector
Time 5
Time 4
35
MS-MS-MS (MSn) of a Fragment ( ) in an Ion
trap
Source
Time 1
Time 3
Time 2
Select one m/z
Dissociate
Select one m/z
Detector
Time 5
Time 4
36
MS-MS-MS (MSn) of a Fragment ( ) in an Ion
trap
Source
Time 1
Time 3
Time 2
Select one m/z
Dissociate
Select one m/z
Dissociate
Detector
Time 5
Time 4
37
MS-MS-MS (MSn) of a Fragment ( ) in an Ion
trap
Source
Time 1
Time 3
Time 2
Select one m/z
Dissociate
Select one m/z
Dissociate
Scan Products
Detector
Time 5
Time 4
38
MS-MS-MS of a fragment from the Peptide
y3 fragment 336.2
205
205
177
177
39
Product Ion Scans may be Software Controlled
  • Can collect MS/MS spectra for complex mixtures
  • 2 examples 1) HPLC-Ion Trap, 2) HPLC-Q-TOF
  • Peptides separated by HPLC
  • HPLC linked directly to analyzer by ESI source
  • Mass analyzer collects continuous MS spectra
  • At pre-determined intensity of a precursor ion,
    MS/MS spectra acquired

40
Ion Current over 60 min
41
Ion Current over 60 min
42
Ion Current over 60 min
MS
43
Ion Current over 60 min
MS
44
Ion Current over 60 min
MS/MS
MS
45
Q-TOF Schematic
Benefits Higher resolution mass accuracy All
ions recorded in parallel
Ref Chemushevich, 2001
46
HPLC MS MS/MS (Q-TOF)Software Controlled
Product Ion Scan
Total Ion Chromatogram
MS of peak eluting at 33.27 min.
MS-MS of m/z 903.5
Ref Chemushevich, 2001
47
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

48
Precursor Ion Scan
  • Produces a spectrum that is an array of precursor
    ions that produce a given product ion.
  • Screening for compound types
  • Compounds that all provide the same fragment ion
    m/z
  • Q1 is scanned
  • These precursor ions collide with target gas (in
    CID)
  • Fragments (product ions) are formed
  • Q3 allows transmission of one fragment ion m/z
  • Software produces spectrum of compounds which
    formed the selected fragment ion.

49
Precursor Ion Scan Detection of
Scan Precursors
(sequential rf/dc)
50
Precursor Ion Scan Detection of
Dissociate
rf/dc 1
(collide with gas)
51
Precursor Ion Scan Detection of
Select fragment
(fixed rf/dc )
52
Precursor Ion Scan Detection of
Dissociate
rf/dc 2
(collide with gas)
53
Precursor Ion Scan Detection of
Select fragment
(fixed rf/dc )
54
Precursor Ion Scan Detection of
Dissociate
rf/dc 3
(collide with gas)
55
Precursor Ion Scan Detection of
Select fragment
(fixed rf/dc )
56
Precursor Ion SpectrumReconstructed by software
Software stores memory of the rf/dc voltages that
coincide with fragments striking the detector!
Q1 rf/dc 3
Q1 rf/dc 2
These rf/dc voltages equal specific m/z values
57
Precursor Ion Scan in Combinatorial Chemistry
  • Combinatorial libraries result from the
    simultaneous synthesis of a great number of
    compounds.
  • analytical challenge to characterize
  • Purpose Determine purity and identity of pooled
    library
  • QQQ mass spectrometer

Ref Triolo, 2001
58
Precursor Ion Scan in Combinatorial Chemistry
  • Library synthesized consisting of fixed (X, Y, Z)
    and variant (amino acids) as
  • If aa1 Arg, then a fragment of m/z 455 is
    formed
  • mass difference due to aa2 provides identity of
    the compound

X-aa1-Y-aa2-Z
Ref Triolo, 2001
59
Precursor Ion Scan of m/z 455 of a pooled library
MS
Precursor Scan
Ref Triolo, 2001
60
Precursor ion Scan for drug metabolites (Q-TOF)
  • Search for metabolites of monoacetylmorphine
    (MAM)
  • Purpose increase specificity of ion selection
  • 7 common fragments identified
  • these fragments used for precursor ion scan
  • Urine sample from subject exposed to MAM
  • During LC run, mass spectrometer switched
  • 2 sec precursor ion scans
  • 1 sec product ion scan

Ref Chemushevich, 2001
61
Q-TOF Schematic
Benefits Higher resolution mass accuracy All
ions recorded in parallel
Ref Chemushevich, 2001
62
Precursor ion Scan for drug metabolites (Q-TOF)
Ion Chromatogram (combined precursor and MS-MS
scans)
Overlaid scans Precursors of multi fragments
common to the metabolites
Example of MS-MS monoacetylmorphine recorded
after detecting m/z 328
Ref Chemushevich, 2001
63
Applying Scan Modes to Peptide Phosphorylation
Mapping
  • Recall Carr 1996
  • QQQ mass spectrometer and precursor ion scanning
    of 79 amu used to detect loss of PO3- from
    phosphopeptides.
  • Sequenced by sequential selection and product ion
    scan
  • NEW HYBRID INSTRUMENT Unique Scanning
    Capabilities

2003
AB 4000 Q TRAP
Le Blanc et al., Proteomics 2003, 3 859-869
64
AB 4000 Q TRAP
  • Hybrid triple quadrupole-linear ion trap mass
    spectrometer
  • QQQ features and LTQ advantages
  • Fast switching times -/

Le Blanc et al., Proteomics 2003, 3 859-869
65
Precursor Ion 79 scan (-)
  • Phosphopeptides lose 79 (PO3) negative
  • Phospho-Tyrosine loses 216 positive
  • Phospho-Serine/Threonine lose 98 (H3PO4) neutral
    (49 amu loss if 2)

Enhanced Resolution ()
Product Ion Scan () of m/z 625.13
Le Blanc et al., Proteomics 2003, 3 859-869
66
Neutral ion loss 49 ()
Survey scan shows 831 amidst other co-eluting
peptides
Product Ion Scan () of m/z 830.9
67
Learning Check Precursor Ion Scan
  • Consider identification of a mixture of
    halogenated compounds by MS-MS
  • Describe a Precursor Ion Scan that might be used
    to identify all monohalogenated benzenes in a
    sample
  • What is the m/z that hits the detector?
  • What happens in Q1, q2, Q3?
  • Draw the spectrum

C 12 Cl 35/37
H 1 Br 79/81
F 19 I 127
68
Learning Check Precursor Ion Scan
  • What m/z hits the detector?
  • What happens in Q1, q2, Q3?
  • Draw the
  • spectrum

Q1
q2
Q3
Relative Intensity
m/z
69
Learning Check Precursor Ion Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Relative Intensity
m/z
70
Learning Check Precursor Ion Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Scan all ions
sequential CID
Fix m/z 77
Relative Intensity
m/z
71
Learning Check Precursor Ion Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Scan all ions
sequential CID
Fix m/z 77
96
112
156/158
204
Relative Intensity
114
m/z
72
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

73
Neutral Loss Scan
  • Produces a spectrum that is an array of ions that
    undergo a common loss (such as loss of H2O)
  • Screen for molecule types
  • Q1 and Q3 are both scanned
  • Q3 is offset by the neutral loss selected
  • The precursor ion collides with target gas (in
    CID)
  • Fragments (product ions) are formed
  • Only compounds providing the selected loss are
    detected

74
Neutral Loss Scan Loss of m/z
Scan Precursors
(sequential rf/dc)
75
Neutral Loss Scan Loss of m/z
Dissociate
rf/dc 1
(collide with gas)
76
Neutral Loss Scan Loss of m/z
Scan for offset m/z
(Offset rf/dc)
77
Neutral Loss Scan Loss of m/z
Dissociate
rf/dc 2
(collide with gas)
78
Neutral Loss Scan Loss of m/z
Scan for offset m/z
(Offset rf/dc)
79
Neutral Loss Scan Loss of m/z
Dissociate
rf/dc 3
(collide with gas)
80
Neutral Loss Scan Loss of m/z
Scan for offset m/z
(Offset rf/dc)
81
Neutral Loss SpectrumReconstructed by software
Software stores memory of the rf/dc voltages that
coincide with fragments striking the detector!
Q1 offset rf/dc 2
The rf/dc voltages equals a specific m/z value
82
Neutral Loss in Newborn Screening for
DiseaseExample shown here Phenylketonuria
  • Many inherited diseases characterized by
    increased levels of certain amino acids in blood
  • Purpose fast, accurate disease screening
  • analysis of dried filter paper samples
  • 2 min. per sample
  • MS-MS Detection lowered false-positives by
    10-fold
  • Extracted sample derivatized to butyl esters
  • Phenylketonuria increased phenylalanine
  • Determined by ratios of amino acids
  • phenylalanine tyrosine
  • Ref Chace, 2001

83
Product Ion Scan of Phenylalanine butyl ester
shows loss of m/z 102
Ref Chace, 2001
84
Control
Blood sample from Normal Newborn
Masses of deuterated internal standards are
underlined
85
Control
Blood sample from Normal Newborn
Masses of deuterated internal standards are
underlined
Phenylketonuria
Blood sample from newborn diagnosed with
phenylketonuria
86
Learning Check Neutral Loss Scan
  • Consider identification of a mixture of
    halogenated compounds by MS-MS
  • Describe a Neutral Loss Scan that might be used
    to identify all Chlorine containing compounds
  • What is the m/z that hits the detector?
  • What happens in Q1, q2, Q3?
  • Draw the spectrum

C 12 Cl 35/37
H 1 Br 79/81
F 19 I 127
87
Learning Check Neutral Loss Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

Q1
q2
Q3
Relative Intensity
m/z
88
Learning Check Neutral Loss Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Relative Intensity
m/z
89
Learning Check Neutral Loss Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
scan offset 35 amu
Scan all ions
sequential CID
Relative Intensity
m/z
90
Learning Check Neutral Loss Scan
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
scan offset 35 amu
Scan all ions
sequential CID
112
Relative Intensity
m/z
91
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

92
Selected Reaction Monitoring (SRM or MRM)
  • Single (SRM) or Multiple (MRM) reaction
    monitoring
  • Quantitative target analyte scan
  • Q1 is fixed to allow transmission of one
    precursor m/z
  • This precursor ion collides with target gas (in
    CID)
  • Fragments (product ions) are formed
  • Q3 is fixed to allow transmission of one fragment
    m/z

93
Selected Reaction Monitoring
Select one m/z
(fixed Vac/Vdc)
94
Selected Reaction Monitoring
Dissociate
(collide with gas)
95
Selected Reaction Monitoring
Select one m/z
(Fixed rf/dc)
96
MRM used to Improve Sensitivity for
Corticosteroid Detection
  • Used illegally as growth promoters in cattle
  • Purpose detect low residue levels in biological
    matrices
  • enhance specificity and sensitivity
  • QQQ mass spectrometer
  • Studied fragmentation of corticosteroids by CID
  • Determined negative mode to produce more specific
    ions
  • Evaluated 3 acquisition methods in negative mode
  • Product ion (Q1 fixed for M acetate-
  • Neutral loss, Loss of 90 amu, (acetic acid plus
    formaldehyde)
  • Multiple reaction monitoring (MRM) (Alternate -90
    and M-H-)

Ref Antignac, 2000
97
Improving Sensitivity for Corticosteroid Detection
MS/MS Product Ion Spectra
betamethasone
Ref Antignac, 2000
98
Product Ion Scan
Select
Dissociate
Scan
99
Comparison Product Ion, Neutral Loss, MRM
Total ion current Chromatograms
1 ng
Ref Antignac, 2000
100
Neutral Loss Scan Loss of m/z
rf/dc 1
rf/dc 2
rf/dc 3
Scan Dissociate Select offset m/z
101
Comparison Product Ion, Neutral Loss, MRM
Neutral Loss 10X more sensitive than MS/MS
100 pg
Ref Antignac, 2000
102
Selected Reaction Monitoring
Select
Dissociate
Select
103
Comparison Product Ion, Neutral Loss, MRM
MRM 10X more sensitive than N.Loss
10 pg
Ref Antignac, 2000
104
Comparison Product Ion, Neutral Loss, MRM
Total ion current Chromatograms
1 ng
Neutral Loss 10X more sensitive than MS/MS
100 pg
MRM 10X more sensitive than N.Loss
10 pg
blank
Ref Antignac, 2000
105
MRM Applied to Mapping Phosphorylation
  • First LC-MS/MS performed to identify protein
  • Theoretical digest /- phosphorylation performed
    to give list of expected peptide ions
  • Instrument software used to link phosphorylated
    parent peptide ions to fragment ions most
    indicative of modification

106
j
MS/MS
Predicted Fragment Mass 1
Peptide w/ potential phosphorylated residue
Predicted Fragment Mass 2
MH
107
Predictive MRM MRM Triggered Information
Dependent Acquisition Identifies and/or Confirms
PTMs
Theoretical Protein Digest, Predicted MRM
Transitions for PO4 Peptides
()
()
Automatic MRM Method Construction
108
Predictive MRM MRM Triggered Information
Dependent Acquisition Identifies and/or Confirms
PTMs
AVDGYVKPQIK
y6
Zoom in
y4
AVDGpYVKPQIK MRM 1
AVDGYVKPQIK
y6
y9
AVDGpYVKPQIK MRM 2
AVDGpYVKPQIK MRM 3
y7
Multiple Transitions Per Peptide
109
Learning Check Selected Ion Monitoring
  • Consider identification of a mixture of
    halogenated compounds by MS-MS
  • Describe a SIM Scan that might be used to
    identify fluorobenzene
  • What is the m/z that hits the detector?
  • What happens in Q1, q2, Q3?
  • Draw the spectrum

C 12 Cl 35/37
H 1 Br 79/81
F 19 I 127
110
Learning Check Selected Ion Monitoring
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

Q1
q2
Q3
Relative Intensity
m/z
111
Learning Check Selected Ion Monitoring
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Relative Intensity
m/z
112
Learning Check Selected Ion Monitoring
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Fix m/z 96
CID
Fix m/z 77
Relative Intensity
m/z
113
Learning Check Selected Ion Monitoring
  • What m/z hits the detector?
  • What happens in Q1 q2 Q3?
  • Draw the
  • spectrum

m/z 77
Q1
q2
Q3
Fix m/z 96
CID
Fix m/z 77
96
Relative Intensity
m/z
114
Scan Modes
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • May be data dependent
  • Precursor ion scan Scan
    Dissociate Select
  • Neutral Loss scan Scan
    Dissociate Scan
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Ion-Molecule reactions Select React
    Scan

115
Ion-Molecule or Ion-Ion Reactions
  • Gas phase reactions
  • Ion-molecule reactions include H/D exchange
  • substitution of deuterium for hydrogen on analyte
  • Ion-Ion reactions include stripping of protons
  • React cation analytes with anions that remove
    charge
  • Q1 allows transmission of one m/z
  • This precursor ion reacts with reagent gas
  • Reaction products are formed
  • Reaction products are scanned through Q3

116
Tandem in Time (Ion Trap) Reaction product Scan
117
Tandem in Time (Ion Trap) Reaction product Scan
Select one m/z
(Fixed rf/dc)
118
Tandem in Time (Ion Trap) Reaction product Scan
React
(extend time in gas)
119
Tandem in Time (Ion Trap) Reaction product Scan
Scan Products
(scan rf/dc)
120
Ion-Ion Reactions Reduce Charge State (Ion Trap)
  • Determine mixtures of relatively high-mass
    proteins
  • (and other types of high-mass biopolymers)
  • Purpose improve resolution of ions produced by
    ESI
  • Custom instrument at Oak Ridge National
    Laboratory
  • Increased mass range over standard ion traps
  • Glow discharge ion source produces singly charged
    anions
  • Used standard proteins to demonstrate method

Ref Stephenson, 1996
121
Ion-Ion reactions reduce protein charge states
Ribonuclease B (14,899 Da ) with family of
N-glycans labelled (m/z scale 1200 - 2200)
Ref Stephenson, 1996
122
Ion-Ion reactions reduce protein charge states
Reaction with C8F15- and C7F13- anions derived
from PDCH perfluoro-1,3-dimethylcyclohexane
9 to 7 charge states reduced to 6 to 3
charge states (m/z scale 1600 - 7200)
Ref Stephenson, 1996
123
Ion-Ion reactions reduce protein charge states
Mixture of Proteins ubiquitin bovine serum
albumin bovin transferrin (m/z scale 700 - 2400)
Ref Stephenson, 1996
124
Ion-Ion reactions reduce protein charge states
295 ms reaction with PDCH (m/z scale 5000 -
24000)
Ref Stephenson, 1996
125
Ion trap MS/MS versus H/D Exchange
126
Ion trap MS/MS versus H/D Exchange
127
Ion-Molecule Reactions in an Ion Trap
  • Minor instrument modification
  • Provides information to compare related compounds
  • Relay mechanism of H/D exchange

Campbell, Rodgers, Marzluff, Beauchamp, J.Am.
Chem.Soc. (1995) 11712840-12854
128
AAA reaction with D2O versus AAAAAAAA has
weaker intramolecular interactions
MH D0
D5
AAA
AAAAA
D6
MH D0
D4
129
H/D exchange of AAAAA vs. acetyl-AAAAA
130
H/D exchange of AAAAA vs. acetyl-AAAAA
131
Finnigan LCQ Ion Trap, Standard configuration
132
Trap modification for ion-molecule reactions
Adapted from Gronert, S. J.Am. Soc. Mass
Spectrom. (1998) 9 845-848
133
Scan Modes Summary
Tandem in Space
Tandem in Time
  • Product ion scan Select
    Dissociate Scan
  • Qualitative Structural Information
  • Precursor ion scan Scan
    Dissociate Select
  • Screen for compound types that lose a detectable
    fragment
  • Neutral Loss scan Scan
    Dissociate Scan
  • Screen for compound types that lose a neutral
  • Selected Reaction Select
    Dissociate Select monitoring (SRM or MRM)
  • Increase sensitivity
  • Ion-Molecule reactions Select React
    Scan
  • Reduce charge state, probe gas phase structure

134
Suggested Reading List References
  • Precursor Ion Scan
  • Triolo A, Altamura, M., Cardinali, F., Sisto, A.,
    Maggi C., Mass spectrometry and combinatorial
    chemistry a short outline, JMS, 2002
    361249-1259.
  • Chemushevich, I.V., Loboda A.V., Thomson B.A., An
    introduction to quadrupole time-of-flight mass
    spectrometry, JMS, 2001, 36849-865.
  • Neutral Loss Scan
  • Chace, D.H., Mass Spectrometry in the Clinical
    Laboratory, Chem. Rev., 2001, 101, 445-477.
  • Multiple Reaction Monitoring
  • Antignac, J.P., Bizec, B.L., Monteau, F.,
    Poulain, F., Andre, F., Collision-induced
    dissociation of corticosteroids in electrospray
    tandem mass spectrometry and development of a
    screening method by high performance liquid
    chromatography/tandem mass spectrometry, RCMS,
    2000, 14, 33-39.

135
Suggested Reading List References (2)
  • Peptide Phosphorylation
  • Carr, S., Huddleston, M.J., Annan, R., Selective
    Detection and Sequencing of Phosphopeptides at
    the Femtomole Level by Mass Spectrometry, Anal.
    Biochem., 1996, 239, 180-192
  • LeBlanc, J.C.Y., Hager, J.W., Ilisiu, A.M.P.,
    Hunter, C., Zhong, F., Chu, I., Unique scanning
    capabilities of a new hybrid linear ion trap mass
    spectrometer (Q TRAP) used for high sensitivity
    proteomics applications, Proteomics, 2003, 3 (6)
    859-869.
  • Hansen, B.T., Jones, J.A., Mason, D.E., Liebler,
    D.C., SALSA A pattern recognition algorithm to
    detect electrophile-adducted peptides by
    automated evaluation of CID spectra in LC-MS-MS
    analyses, Analytical Chem., 2001, 73 (8)
    1676-1683.
  • Ion-Molecule Reactions
  • Stephenson, J.L., McLuckey, S A., Ion/Ion Proton
    Transfer Reactions for Protein Mixture Analysis,
    Anal. Chem, 1996, 68, 4026-4032.
  • Campbell, S., Rodgers, M.T., Marzluff, E.M.,
    Beauchamp, J.L., Deuterium exchange reactions as
    a probe of biomolecule structure. Fundamental
    studies of gas phase H/D exchange reactions of
    protonated glycine oligomers with D2O, CD3OD,
    CD3CO2D, and ND3, J.Am.Chem.Soc., 1995, 117 (51)
    12840-12854
  • Gronert, S., Estimation of effective ion
    temperatures in a quadrupole ion trap, JASMS,
    1998, 9 (8) 845-848.
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