Transcript MS-MS Scan Modes - University of Arizona

MS/MS Scan Modes
Linda Breci
Chemistry Mass Spectrometry Facility
University of Arizona
MS Summer Workshop
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)
Analyzers
TOF
Quadrupole
Quadrupole Ion trap
FTICR
Magnetic sector
Quadrupole (Q)
Continuous source:
One m/z value could
be observed continuously
http://www-methods.ch.cam.ac.uk/meth/ms/theory/quadrupole.html
Time-of-Flight (TOF)
field-free drift zone
http://www.kore.co.uk/MS-200_principles.htm
TOF
m/z
V
KE = eV = ½mv2
Ions collected
v = D/t
or lost during
previous ½m(D/t)
TOF 2 = eV
pulse
1/ 2


t= m  D
 2zeV 
Detector
D
m = mass
V = velocity
D = distance of flight
t = time of flight
KE = kinetic energy
e = charge
Quadrupole Ion Trap (QIT)
Voltage gate “shut”
during scan of
trapped ions
http://www-methods.ch.cam.ac.uk/meth/ms/theory/iontrap.html
Fourier Transform-Ion Cyclotron
Resonance (FTICR)
Voltage gate “shut”
during scan of
trapped ions
http://www-methods.ch.cam.ac.uk/meth/ms/theory/iontrap.html
Analyzers based on magnetic fields: Sector
Magnetic forces move ions in a circular path
Continuous source:
One m/z value could
be observed continuously
demo
http://www.casetechnology.com/implanter/magnet.html
Quadrupole (Q)
Continuous source:
One m/z value could
be observed continuously
http://www-methods.ch.cam.ac.uk/meth/ms/theory/quadrupole.html
Tandem in Space vs. Tandem in Time MS/MS
• Space (Analyzers cannot trap, must be linked)
– Triple Quad
Q1
q2 (gas)
Q3
– TOF
TOF
(gas)
TOF
– Sector
Sector
(gas)
Sector
• Many combinations of linked analyzers possible
• Time (Trapping analyzers)
– Quadrupole Ion trap
– ICR
Time 1
• Most activation by CID (CAD)
Time 2
Time 3
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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
Quadrupole (Q) Single analyzer scan
Source
Q1
Detector
Quadrupole (Q) Single analyzer scan
Q1
Source
(1st Vac/Vdc)
Detector
Quadrupole (Q) Single analyzer scan
Q1
Source
(2nd Vac/Vdc)
Detector
Quadrupole (Q) Single analyzer scan
Q1
Source
(3rd Vac/Vdc)
Detector
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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
Tandem in Space (QQQ) – Product Ion Scan
Source
Q1
Select one m/z
(fixed Vac/Vdc)
(gas)
Q3
Detector
Tandem in Space (QQQ) – Product Ion Scan
Source
Q1
(gas)
Dissociate
(collide with gas)
Q3
Detector
Tandem in Space (QQQ) – Product Ion Scan
Source
Q1
(gas)
Q3
Scan Products
(scan Vac/Vdc)
Detector
Tandem in Time (Ion Trap) -- Product Ion Scan
Source
Time 1
Time 2
Time 3
Detector
Tandem in Time (Ion Trap) -- Product Ion Scan
Source
Time 1
Select one m/z
Time 2
Time 3
Detector
Tandem in Time (Ion Trap) -- Product Ion Scan
Source
Time 1
Time 2
Time 3
Dissociate
(collide with gas)
Detector
Tandem in Time (Ion Trap) -- Product Ion Scan
Source
Time 1
Time 2
Time 3
Scan Products
Detector
MS of a Peptide (
Source
Time 1
) in an Ion trap
Time 2
Time 3
Detector
• Scan = Voltage Ramped
• Sequential m/z hit detector
MS of a Peptide (YGGFL
)
556.2
100
O
80
Relative Intensity
H2N
CH
CH2
C
O
H
N
CH
C
H
O
H
N
CH
C
O
H
N
H
CH
CH2
60
C
O
H
N
CH
C
OH
CH2
CH
CH3
CH3
40
OH
20
0
200
300
400
m/z
500
600
MS of a Peptide (YGGFL)
Select for MS-MS
(YGGFL Parent)
100
O
80
Relative Intensity
H2N
CH
CH2
C
O
H
N
CH
C
H
O
H
N
CH
C
556.2
O
H
N
H
CH
CH2
60
C
O
H
N
CH
C
OH
CH2
CH
CH3
CH3
40
OH
20
0
200
300
400
m/z
500
600
MS-MS of a Peptide (
Source
Time 1
Select one m/z
Time 2
Dissociate
) in an Ion trap
Time 3
Scan Products
Detector
MS-MS of a Peptide (YGGFL, m/z = 556.2)
425
100
556.2
Relative Intensity
80
60
40
538
397
20
279
336
0
200
300
400
m/z
500
600
MS-MS of a Peptide (YGGFL, m/z = 556.2)
b4
425
b4
100
YGGFL
y3
Relative Intensity
80
y2
556.2
60
40
a4
397
20
y2
279
-H2O
538
y3
336
0
200
300
400
m/z
500
600
MS-MS of a Peptide (YGGFL, m/z = 556.2)
b4
425
b4
100
YGGFL
y3
Relative Intensity
80
y2
556.2
60
Select for MS-MS-MS
(YGGFL Fragment)
40
a4
397
20
y2
279
-H2O
538
y3
336
0
200
300
400
m/z
500
600
MS-MS-MS (MSn) of a Fragment (
Source
Time 1
Select one m/z
Time 2
) in an Ion trap
Time 3
Dissociate
Time 4
Time 5
Detector
MS-MS-MS (MSn) of a Fragment (
Source
Time 1
Select one m/z
Time 2
Dissociate
Time 4
) in an Ion trap
Time 3
Select one m/z
Time 5
Detector
MS-MS-MS (MSn) of a Fragment (
Source
Time 1
Select one m/z
Time 2
Dissociate
) in an Ion trap
Time 3
Select one m/z
Dissociate
Time 4
Time 5
Detector
MS-MS-MS (MSn) of a Fragment (
Source
Time 1
Select one m/z
Time 2
Dissociate
) in an Ion trap
Time 3
Select one m/z
Dissociate
Scan Products
Time 4
Time 5
Detector
MS-MS-MS of a fragment from the Peptide
100
205
y3 fragment
336.2
205
177
Relative Intensity
80
O
H2N
60
CH
C
O
H
N
H
CH
C
O
H
N
CH2
CH
CH2
CH
40
CH3
20
177
0
200
300
400
m/z
500
C
600
CH3
OH
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
Ion Current
over 60 min
Ion Current
over 60 min
Ion Current
over 60 min
MS
Ion Current
over 60 min
MS
Ion Current
over 60 min
MS/MS
MS
Q-TOF Schematic
Benefits:
Higher resolution & mass accuracy
All ions recorded in parallel
Ref: Chemushevich, 2001
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
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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.
Precursor Ion Scan – Detection of
Source
Q1
Scan Precursors
(sequential rf/dc)
(gas)
Q3
Detector
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Dissociate
rf/dc 1
(collide with gas)
Q3
Detector
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Select fragment
(fixed rf/dc
)
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Dissociate
rf/dc 2
(collide with gas)
Q3
Detector
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Select fragment
(fixed rf/dc
)
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Dissociate
rf/dc 3
(collide with gas)
Q3
Detector
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Select fragment
(fixed rf/dc
)
Precursor Ion Spectrum
Reconstructed by software
Software stores memory of the rf/dc voltages that coincide
with fragments striking the detector!
100
Q1 rf/dc 3
Relative Intensity
80
Q1 rf/dc 2
60
These rf/dc voltages
equal specific
m/z values
40
20
0
200
300
400
m/z
500
600
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
Precursor Ion Scan in Combinatorial Chemistry
• Library synthesized consisting of fixed (X, Y, Z) and
variant (amino acids) as:
X-aa -Y-aa -Z
1
2
• If aa1 = Arg, then a fragment of m/z 455 is formed
455
If aa1 = Arg:
X-Arg-Y-aa2-Z
• mass difference due to aa2 provides identity of the
compound
Ref: Triolo, 2001
Precursor Ion Scan of m/z 455 of a pooled library
MS
Precursor Scan
Ref: Triolo, 2001
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
Q-TOF Schematic
Benefits:
Higher resolution & mass accuracy
All ions recorded in parallel
Ref: Chemushevich, 2001
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
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
2003
• NEW HYBRID INSTRUMENT: Unique Scanning
Capabilities
AB 4000 Q TRAP
Le Blanc et al., Proteomics 2003, 3: 859-869
AB 4000 Q TRAP
• Hybrid triple quadrupole-linear ion trap mass
spectrometer
• QQQ features and LTQ advantages
• Fast switching times -/+
Precursor Ion 79 scan (-)
-/+
Polarity Switch
700 ms
Enhanced Resolution (+)
Product Ion Scan (+)
Cycle time
< 4.5 s
Add to
Exclusion
list
-/+
Polarity Switch
700 ms
Le Blanc et al., Proteomics 2003, 3: 859-869
Precursor Ion 79 scan (-)
Enhanced
Resolution (+)
• Phosphopeptides lose
79 (PO3): negative
• Phospho-Tyrosine
loses 216: positive
• PhosphoSerine/Threonine lose
98 (H3PO4): neutral
(49 amu loss if 2+)
Product Ion Scan (+)
of m/z 625.13
Le Blanc et al., Proteomics 2003, 3: 859-869
Neutral ion loss 49 (+)
Survey scan shows 831 amidst
other co-eluting peptides
Product Ion Scan (+)
of m/z 830.9
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
F
Br
I
Cl
C
12 Cl
35/37
H
1 Br
79/81
F
19 I
127
Learning Check: Precursor Ion Scan
• What m/z hits the detector?
• What happens in Q1, q2, Q3?
Q1
q2
• Draw the
spectrum
Q3
Relative
Intensity
0
50
100
m/z
150
200
Learning Check: Precursor Ion Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
Q3
Relative
Intensity
0
50
100
m/z
150
200
+
Learning Check: Precursor Ion Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
sequential CID
Scan all ions
• Draw the
spectrum
Q3
Fix: m/z 77
Relative
Intensity
0
50
100
m/z
150
200
+
Learning Check: Precursor Ion Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
Q3
sequential CID
Scan all ions
Fix: m/z 77
96 112
• Draw the
spectrum
Relative
Intensity
+
156/158
204
114
0
50
100
m/z
150
200
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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
Neutral Loss Scan Loss of m/z =
Source
Q1
Scan Precursors
(sequential rf/dc)
(gas)
Q3
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Dissociate
rf/dc 1
(collide with gas)
Q3
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Scan for offset m/z
(Offset rf/dc)
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Dissociate
rf/dc 2
(collide with gas)
Q3
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Scan for offset m/z
(Offset rf/dc)
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Dissociate
rf/dc 3
(collide with gas)
Q3
Detector
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Scan for offset m/z
(Offset rf/dc)
Detector
Neutral Loss Spectrum
Reconstructed by software
Software stores memory of the rf/dc voltages that coincide
with fragments striking the detector!
100
Q1 offset rf/dc 2
Relative Intensity
80
The rf/dc voltages
equals a specific
m/z value
60
40
20
0
200
300
400
m/z
500
600
Neutral Loss in Newborn Screening for Disease
Example 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
Product Ion Scan of Phenylalanine butyl ester
shows loss of m/z 102
Ref: Chace, 2001
Control
Blood sample from
Normal Newborn
Masses of deuterated internal
standards are underlined
Control
Blood sample from
Normal Newborn
Masses of deuterated internal
standards are underlined
Phenylketonuria
Blood sample from
newborn diagnosed
with phenylketonuria
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
F
Br
I
Cl
C
12 Cl
35/37
H
1 Br
79/81
F
19 I
127
Learning Check: Neutral Loss Scan
• What m/z hits the detector?
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
Q3
Relative
Intensity
0
50
100
m/z
150
200
Learning Check: Neutral Loss Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
+
Q3
Relative
Intensity
0
50
100
m/z
150
200
Learning Check: Neutral Loss Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
Q3
sequential CID
Scan all ions
+
scan offset
35 amu
Relative
Intensity
0
50
100
m/z
150
200
Learning Check: Neutral Loss Scan
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
Q3
sequential CID
Scan all ions
+
scan offset
35 amu
112
• Draw the
spectrum
Relative
Intensity
0
50
100
m/z
150
200
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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
Selected Reaction Monitoring
Source
Q1
Select one m/z
(fixed Vac/Vdc)
(gas)
Q3
Detector
Selected Reaction Monitoring
Source
Q1
(gas)
Dissociate
(collide with gas)
Q3
Detector
Selected Reaction Monitoring
Source
Q1
(gas)
Q3
Select one m/z
(Fixed rf/dc)
Detector
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
Improving Sensitivity for Corticosteroid Detection
MS/MS Product Ion Spectra
betamethasone
Ref: Antignac, 2000
Product Ion Scan
Source
Q1
Select
(gas)
Dissociate
Q3
Scan
Detector
Comparison: Product Ion, Neutral Loss, MRM
1 ng
Total ion current
Chromatograms
Ref: Antignac, 2000
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
rf/dc 1
rf/dc 2
rf/dc 3
Scan
Dissociate
Select offset m/z
Detector
Comparison: Product Ion, Neutral Loss, MRM
100 pg
Neutral Loss
10X more
sensitive
than MS/MS
Ref: Antignac, 2000
Selected Reaction Monitoring
Source
Q1
Select
(gas)
Dissociate
Q3
Select
Detector
Comparison: Product Ion, Neutral Loss, MRM
10 pg
MRM
10X more
sensitive
than N.Loss
Ref: Antignac, 2000
Comparison: Product Ion, Neutral Loss, MRM
1 ng
Total ion current
Chromatograms
100 pg
Neutral Loss
10X more
sensitive
than MS/MS
10 pg
MRM
10X more
sensitive
than N.Loss
blank
Ref: Antignac, 2000
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
j
MS/MS
Peptide w/ potential
phosphorylated
residue
Predicted
Fragment
Mass 1
Predicted
Fragment
Mass 2
[M+H]+
k
MS/MS
Peptide w/ potential
phosphorylated
residue
[M+H]+
Predicted
Fragment
Mass 1
Predicted
Fragment
Mass 2
Predicted
Fragment
Mass 3
Predictive MRM & MRM Triggered Information
Dependent Acquisition Identifies and/or Confirms
PTM’s
Theoretical Protein Digest, Predicted MRM Transitions for PO4 Peptides
(…)
(…)
Automatic MRM Method Construction
Predictive MRM & MRM Triggered Information
Dependent Acquisition Identifies and/or Confirms
PTM’s
AVDGYVKPQIK
y6
Zoom in
y4
y9
y7
AVDGpYVKPQIK MRM 1
AVDGpYVKPQIK MRM 2
AVDGpYVKPQIK MRM 3
AVDGYVKPQIK y6
Multiple Transitions Per Peptide
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
F
Br
I
Cl
C
12 Cl
35/37
H
1 Br
79/81
F
19 I
127
Learning Check: Selected Ion Monitoring
• What m/z hits the detector?
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
Q3
Relative
Intensity
0
50
100
m/z
150
200
Learning Check: Selected Ion Monitoring
• What m/z hits the detector?
m/z = 77
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
Q3
Relative
Intensity
0
50
100
m/z
150
200
+
Learning Check: Selected Ion Monitoring
m/z = 77
• What m/z hits the detector?
• What happens in Q1 q2 Q3?
Q1
q2
CID
Fix: m/z 96
• Draw the
spectrum
Q3
Fix: m/z 77
Relative
Intensity
0
50
100
m/z
150
200
+
Learning Check: Selected Ion Monitoring
m/z = 77
• What m/z hits the detector?
• What happens in Q1 q2 Q3?
Q1
q2
Q3
CID
Fix: m/z 96
Fix: m/z 77
96
• Draw the
spectrum
Relative
Intensity
0
50
100
m/z
150
200
+
Scan Modes
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
• Precursor ion scan
Scan
• Neutral Loss scan
Scan
• Selected Reaction
Select
monitoring (SRM or MRM)
• Ion-Molecule reactions Select
Dissociate
Dissociate
Dissociate
Select
Scan
Select
React
Scan
– May be data dependent
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
Tandem in Time (Ion Trap) – Reaction product Scan
Source
Time 1
Time 2
Time 3
Detector
Tandem in Time (Ion Trap) – Reaction product Scan
Source
Time 1
Select one m/z
(Fixed rf/dc)
Time 2
Time 3
Detector
Tandem in Time (Ion Trap) – Reaction product Scan
Source
Time 1
Time 2
Time 3
React
(extend time in gas)
Detector
Tandem in Time (Ion Trap) – Reaction product Scan
Source
Time 1
Time 2
Time 3
Scan Products
(scan rf/dc)
Detector
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
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
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
Ion-Ion reactions reduce protein charge states
Mixture of Proteins:
ubiquitin
bovine serum albumin
bovin transferrin
(m/z scale 700 - 2400)
Ref: Stephenson, 1996
Ion-Ion reactions reduce protein charge states
295 ms reaction with PDCH
(m/z scale 5000 - 24000)
Ref: Stephenson, 1996
Ion trap MS/MS versus H/D Exchange
Ion trap MS/MS versus H/D Exchange
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) 117:12840-12854
AAA reaction with D2O versus AAAAA
AAA has weaker intramolecular interactions
MH+
D0
D5
AAA
D6
MH+
D0
D4
AAAAA
H/D exchange of AAAAA vs. acetyl-AAAAA
H/D exchange of AAAAA vs. acetyl-AAAAA
Finnigan LCQ Ion Trap, Standard configuration
Trap modification for ion-molecule reactions
Adapted from: Gronert, S. J.Am. Soc. Mass Spectrom. (1998) 9: 845-848
Scan Modes Summary
Tandem in Space
Tandem in Time
• Product ion scan
Q1
q2 (gas)
Q3
Time 1
Time 2
Time 3
Select
Dissociate
Scan
Dissociate
Select
– Qualitative Structural Information
• Precursor ion scan
Scan
– 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
monitoring (SRM or MRM)
Dissociate
Select
React
Scan
– Increase sensitivity
• Ion-Molecule reactions
Select
– Reduce charge state, probe gas phase structure
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; 36:1249-1259.
Chemushevich, I.V., Loboda A.V., Thomson B.A., An introduction to quadrupole – time-of-flight
mass spectrometry, JMS, 2001, 36:849-865.
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.
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.