MS/MS Scan Modes
Árpád Somogyi
• Eötvös University, Budapest
April 16, 2012
MS/MS Scan Modes
Product Ion Scan
Select
Dissociate
Scan
Scan
Dissociate
Select
Precursor Ion Scan
Neutral Loss Scan
Δ
Scan
Dissociate
Scan
Select
Dissociate
Select
Selected Reaction
Monitoring (SRM)
1
Scan modes in a triple quadrupole (QqQ)
(one quadrupole shown here)
http://www-methods.ch.cam.ac.uk/meth/ms/theory/quadrupole.html
Scan
300
Voltage
Analyte
Mixture
200
RF
DC
100
100
200
300
Vm1
m/zm1
Vm2
Vm3
m/zm2 m/zm3 mass spectrum
2
Scan
300
Voltage
200
DC
100
Analyte
Mixture
RF
100
200
300
Vm1
Vm2
Vm3
100
300100
100
300
200200 300
100
200 300
200
Scan
300
Voltage
Analyte
Mixture
200
RF
DC
100
100
200
300
Vm1
Vm2
Vm3
100
300100
100
300
200200 300
100
200 300
200
3
Scan
RF
300
Voltage
200
DC
100
Analyte
Mixture
100
200
300
Vm1
Vm2
Vm3
100
300100
100
300
200200 300
100
200 300
200
Select
Voltage
200
Analyte
Mixture
RF
DC
100
200
300
Vm2
Desired
Analyte
m/zm2
mass spectrum
4
Modes of scanning in a Triple Quadrupole (QQQ)
Q1
scan or
select
q2 (gas)
rf only
Q3
scan or
select
• Quadrupole is a mass filter
• QQQ used in this tutorial to describe scan modes
– Q1 and Q3 = analyzers
– q2 (middle quadrupole) used for CID (dissociation)
• Ways to set quadrupoles: Scan, Select & rf only
• Other instruments are used
A variety of instruments are used for MS/MS
To name a few…
5
QQQ
Q3
Q1
q2
Benefits:
Simple, ion filter
Good for quantification
Q-TOF
Q1
q2
TOF
Benefits:
Higher resolution & mass accuracy
All ions recorded in parallel
Ref: Chemushevich, 2001
6
Q-Linear Ion Trap (Q-trap)
q2
Q1
LIT
Benefits:
Quadrupole-like CID spectra with ion trap sensitivity
No ion trap low mass cutoff
Ref: Hopfgartner, 2003
LT-Orbitrap (pictured with ETD source)
quadrupole
Mass
linear
filter
C-trap
Ion trap
Q1
API ion source
orbitrap
Orbi
q2
HCD
collision
cell
reagent
Ion
source
reagent reagent
Benefits:
LTQ: Ion trap sensitivity
Orbi: High dynamic range
& high resolution & mass accuracy
7
Trapping Instruments
Q1
Q3
q2
Benefits:
Sensitivity
MS^n (most)
MS/MS Scan Modes
Product Ion Scan
Select
Dissociate
Scan
Scan
Dissociate
Select
Precursor Ion Scan
Neutral Loss Scan
Δ
Scan
Dissociate
Scan
Select
Dissociate
Select
Selected Reaction
Monitoring (SRM)
8
Product Ion Scan
Q1
Select
q2 (gas)
Q3
Dissociate
Scan
• Qualitative structural information
• 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
Prerequisite: Produce an MS spectrum for selection
Output = MS/MS spectrum
Tandem in Space (QQQ) – Product Ion Scan
Source
Q1
(gas)
Q3
Detector
Select one m/z
(fixed Vac/Vdc)
9
Tandem in Space (QQQ) – Product Ion Scan
Source
Q1
(gas)
Q3
Detector
Dissociate
Scan Products
(collide with gas)
(scan Vac/Vdc)
MS … select … MS/MS
select
MS
MS/MS
10
MS/MS of a Peptide (YGGFL, m/z = 556.2)
b4
425
a/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
500
600
m/z
Multiple stages of MS in a trapping instrument
MSn of trocade (a drug metabolism study)
MS
MS2
MS3
MS4
Ref: Hopfgartner, 2003
11
Product Ion Scans may be Software Controlled
•
•
•
•
•
Goal: collect MS/MS spectra for complex mixtures
Complex mixture can be 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
– Data Dependent acquisition
– Dynamic Exclusion = exclude repeats
26.47
Ion Current
over 60 min
571.29
MS/MS
MS
12
Advantages for product ion scan
Select
Dissociate
Scan
NOTES
QQQ
Q-trap
Q-TOF
TOF-TOF
Ion Trap (3D, LT)
ICR
Q or Trap-ICR
LT-Orbitrap
Orbitrap Animation
13
MS/MS Scan Modes
Product Ion Scan
Select
Dissociate
Scan
Scan
Dissociate
Select
Precursor Ion Scan
Neutral Loss Scan
Δ
Scan
Dissociate
Scan
Select
Dissociate
Select
Selected Reaction
Monitoring (SRM)
Precursor Ion Scan
•
•
•
•
•
•
•
•
Q1
q2 (gas)
Scan
Dissociate
Q3
Select
Screen for precursor ions that produce a given product ion
Q1 is scanned
All precursor ions collide with target gas (in CID)
Fragments (product ions) are formed
Q3 allows transmission of one fragment ion m/z
Run as HPLC-MS/MS experiment
Prerequisite: Determine expected product ions by MS/MS
Output = chromatogram showing time/intensity of precursors
of interest and reconstructed spectrum
14
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Scan Precursors
(sequential rf/dc)
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 1
at rf/dc 1
(collide with gas)
15
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Select fragment
at rf/dc 1
(fixed rf/dc
)
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 2
at rf/dc 2
(collide with gas)
16
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Select fragment
at rf/dc 2
(fixed rf/dc
)
Precursor Ion Scan – Detection of
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 3
at rf/dc 3
(collide with gas)
17
Precursor Ion Scan – Detection of
Q1
Source
(gas)
Q3
Detector
Select fragment
at rf/dc 3
(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
500
600
m/z
18
Precursor ion result – precursor of 436.2
Coming into Q1
In Q1, at one rf/dc ratio, m/z = 842.5
all ions
In mixture
(TIC)
NOT DETECTED
Q3 fixed to detect 436.2
m/z = 842.5
Precursor ion is
Fragmented in q2
Reconstructed chromatogram
m/z = 436.2
total ion
Current
m/z 842.5
hits the detector
Learning Check: FACT SHEET
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
19
Learning Check: PROBLEM SOLVER
Precursor Ion Scan
1) Calculate the mass of one
precursor ion, for example,
fluorobenzene
_____ carbon
@
12 = _____
_____ hydrogen @
1 = _____
_____ fluorine
@
19 = _____
Total = _____
2) Draw a likely fragment ion
common to all of these
analytes? (assume a
simple fragment from M+ is
formed)
3) Calculate the mass of the
common fragment
_____ carbon
@
_____ hydrogen @
12 = _____
1 = _____
Total = _____
Learning Check: PROBLEM SOLVER
Precursor Ion Scan
1) Calculate the mass of one _____
6 carbon @ 12 = _____
72
precursor ion, for example, _____
5 hydrogen @ 1 = _____
5
fluorobenzene
1 fluorine @ 19 = _____
19
_____
96
Total = _____
2) Draw a likely fragment ion
common to all of these
analytes? (assume a
simple fragment from M+ is
formed)
3) Calculate the mass of the
common fragment
.
F+
H
H
H
H
H
_____
6 carbon
@
12 = _____
72
_____
5 hydrogen @
1 = _____
5
Total = _____
77
20
Learning Check: Precursor Ion Scan
.+
H
6 Carbon @ 12 = 72
5 hydrogen @ 1 = 5
H
• What happens in Q1 q2 Q3?
Q1
q2
• Draw the
spectrum
H
Q3
sequential CID
Cl
Br
FI
H
m/z = 77
• What m/z hits the detector?
Scan all ions
H
Fix: m/z 77
96 112
156/158
204
114
Relative
Intensity
77
19
204
77
77+
++127
35
79=
===96
112
156
0
50
100
m/z
150
200
Precursor Ion Scan: A literature example
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
PROBLEM:
MS SCAN IS COMPLEX
AND PROVIDES LITTLE
INFORMATION
Triolo, 2001
21
Precursor Ion Scan: A literature example
Combinatorial Chemistry
• The compound components X, Y, Z are not identified
– mass of X = 299
Library compounds:
– mass of Y = 40
X-AA1-Y-AA2-Z
– mass of Z = 100
• Library compounds, example if AA1 = Arg, AA2 = Ala:
– X-Arg-Y-Ala-Z [mass of Arg = 156, Ala = 71]
– mass: 299 + 156 + 40 + 71 + 100 = 666
– for mass spectrometry, add 1 proton to form ion: 666 + 1 = 667
• When AA1 = Arg, a fragment will form, m/z = 455
m/z = 455
X-Arg-Y-AA2-Z
Triolo, 2001
Learning Check: FACT SHEET
Precursor Ion Scan in Combinatorial Chemistry
Alanine
Arginine
Asparagine
Aspartic Acid
Cystein
Glutamic Acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
ALA
ARG
ASN
ASP
CYS
GLU
GLN
GLY
HIS
ILE
LEU
LYS
MET
PHE
PRO
SER
THR
TRP
TYR
VAL
71
156
114
115
103
129
128
57
137
113
113
128
131
147
97
87
101
186
163
99
R
X
Sum
?
AA1
Y
AA2
Z
299 156
40
?
100
455
140 + ?
595 + AA2 + 1 = Precursor Mass
Library compounds:
X-AA1-Y-AA2-Z
A fragment ion will form for
cleavage at this bond when
aa1 = Arginine
m/z = 455
X-AA1-Y-AA2-Z
22
Learning Check: precursor scan results
Precursor
ARG
[M+H]+
653
667
683
693
695
697
709
710
711
724
725
727
733
743
752
759
AA1
156
156
156
156
156
156
156
156
156
156
156
156
156
156
156
156
X
299
299
299
299
299
299
299
299
299
299
299
299
299
299
299
299
?
Y H+
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
40 1
AA2
Z
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
The precursor ion results of
this experiment are shown
in the left column
find the amino acid
for each of these
compounds
Learning Check: FACT SHEET
Precursor Ion Scan in Combinatorial Chemistry
• Consider identification of a
mixture of these library
compounds by MS/MS
• Describe a Precursor Ion
Scan that might be used to
determine that all amino acids
are represented in position 2
in the compounds (AA2) if
position 1 = Arg
• What is the m/z that hits the
detector?
• What happens in Q1, q2, Q3?
• Draw the spectrum
Library compounds:
X-AA1-Y-AA2-Z
A fragment ion will form for
cleavage at this bond when
aa1 = Arginine
m/z = 455
X-AA1-Y-AA2-Z
23
Learning Check: Precursor Ion Scan in
Combinatorial Chemistry
m/z = 455
• What m/z hits the detector?
X-Arg-+
(299) + (156)
• What happens in Q1, q2, Q3?
Q1
q2
Scan all ions
Q3
sequential CID
Gly
Pro
Fix: m/z 455
Phe
• Draw the
X-Arg-Y-Gly-Z
X-Arg-Y-Pro-Z
X-Arg-Y-Phe-Z
spectrum
(299+156+40+57+100 +1 = 653)
for a few
600
650
700
750
800
compounds
m/z
Precursor Ion Scan of m/z 455 of a pooled library
MS
Precursor Scan
Ref: Triolo, 2001
24
Advantages for precursor scan
Scan
Select
Dissociate
NOTES
QQQ
Q-trap
Q-TOF
TOF-TOF
Ion Trap (3D, LT)
ICR
Q or Trap-ICR
LT-Orbitrap
MS/MS Scan Modes
Product Ion Scan
Select
Dissociate
Scan
Scan
Dissociate
Select
Precursor Ion Scan
Neutral Loss Scan
Δ
Scan
Dissociate
Scan
Select
Dissociate
Select
Selected Reaction
Monitoring (SRM)
25
Neutral Loss Scan
Q1
q2 (gas)
Q3
Δ
Scan
•
•
•
•
•
•
•
•
Dissociate
Scan
(offset from Q1)
Screen for ions that undergo a common loss
Q1 and Q3 are both scanned
Q3 is offset by the neutral loss selected
The precursor ion collides in q2 forming fragments
Compounds providing the selected loss are detected
Run as HPLC-MS/MS experiment
Prerequisite: Determine expected loss by MS/MS
Output = chromatogram showing time/intensity of precursors
of interest and reconstructed spectrum
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Scan Precursors
(sequential rf/dc)
26
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 1
(collide with gas)
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Scan for offset m/z
(Offset rf/dc)
27
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 2
(collide with gas)
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Scan for offset m/z
(Offset rf/dc)
28
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Dissociate
rf/dc 3
(collide with gas)
Neutral Loss Scan Loss of m/z =
Source
Q1
(gas)
Q3
Detector
Scan for offset m/z
(Offset rf/dc)
29
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
500
600
m/z
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
30
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
.+
H
H
H
H
Neutral loss of 35 or 37
m/z of Q1 less 35
• What m/z hits the detector?
for example:
chloro-benzene:112-35 = 77
• What happens in Q1 q2 Q3?
Q1
q2
Q3
scan offset
35 amu
sequential CID
Scan all ions
H
112
Cl +
-35
• Draw the
spectrum
Relative
Intensity
0
50
100
m/z
150
200
31
Neutral Loss Scan: A literature example
Drug Metabolite
• Early stages in design of a drug metabolism study
• Want to “Fish out” relevant metabolites
• Metabolites are in human urine after administration of tolcapone
– tolcapone is a catechol-O-methyl transferase inhibitor
• Possible metabolite is a glucoronide of tolcapone
– metabolites are structurally related to parent drug
– but, product ion spectra may be energy dependent
tolcapone
Hopfgartner, 2003
Neutral Loss Scan: A literature example
Drug Metabolite
researchers expect a metabolite that is a
glucuronide of tolcapone
tolcapone
Mass = 273
Expected conjugate:
mass
273 = tolcapone
176 = glucuronide add’n
449
OH
O
HO
OH
O
HO
OH
tolcapone
glucoronide
Mass = 449
glucuronide
conjugates
commonly provide
mass loss of 176
32
Neutral Loss Scan: A literature example
Q-trap (Q3 = Linear ion trap)
Hopfgartner, 2003
Metabolite of tolcapone:LC-MS/MS Analysis of human urine
TIC of neutral loss
of 176 Da
m/z (Neg ion):
tolcapone
conjugate = 449
loss of H = -1
neutral loss spectrum
at t = 5.8 min
MS/MS spectrum
30 eV
same fragments as
MS/MS of tolcapone
MS/MS spectrum
50 eV
Hopfgartner, 2003
33
Advantages for neutral loss scan
Scan
Dissociate
Scan
NOTES
QQQ
Q-trap
Q-TOF
TOF-TOF
Ion Trap (3D, LT)
ICR
Q or Trap-ICR
LT-Orbitrap
MS/MS Scan Modes
Product Ion Scan
Select
Dissociate
Scan
Scan
Dissociate
Select
Precursor Ion Scan
Neutral Loss Scan
Δ
Scan
Dissociate
Scan
Select
Dissociate
Select
Selected Reaction
Monitoring (SRM)
34
Selected Reaction Monitoring (SRM or MRM)
Q1
Select
•
•
•
•
•
•
•
•
q2 (gas)
Q3
Dissociate
Select
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 in q2 forming fragments
Q3 is fixed to allow transmission of one fragment m/z
Run as HPLC-MS/MS experiment
Prerequisite: Determine expected product ions by MS/MS
Output = chromatogram showing time/intensity of
precursors of interest and reconstructed spectrum
Selected Reaction Monitoring
Source
Q1
(gas)
Q3
Detector
Select one m/z
(fixed Vac/Vdc)
35
Selected Reaction Monitoring
Source
Q1
(gas)
Q3
Detector
Dissociate
(collide with gas)
Selected Reaction Monitoring
Source
Q1
(gas)
Q3
Detector
Select one m/z
(Fixed rf/dc)
36
Learning Check: Selected Ion Monitoring
F
• Consider identification of a
mixture of halogenated
compounds by MS/MS
• Describe a SRM 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
Br
I
Cl
C
12 Cl
35/37
H
1 Br
79/81
F
19 I
127
Learning Check: Selected Ion Monitoring
m/z = 77
H
H
H
• What m/z hits the detector?
H
• What happens in Q1 q2 Q3?
Q1
q2
Q3
CID
Fix: m/z 96
.+
H
Fix: m/z 77
96
F
• Draw the
spectrum
Relative
Intensity
0
50
100
m/z
150
200
37
MRM example: Detection of an antiviral drug and
it’s metabolite in human plasma
• herpes virus replication inhibited by action of acyclovir but
low bioavailability
• valacyclovir metabolizes to acylovir with high bioavailability
• Goal: accurate detection in plasma
• QQQ mass spectrometer, MDS SCIEX API-4000
• Studied fragmentation of compounds by CID
acyclovir (ACV)
fluconazole
(internal std - IS)
valacyclovir (VCV)
Ref: Yadav, 2009
Product ion mass spectra
VCV 325.2/152.2
ACV 226.2/152.2
IS 307.1/220.3
38
MRM chromatograms VCV & IS in plasma
VCV
ACV
IS
blank
IS only
VCV & IS
plasma
VCV & IS
plasma
(subject)
Mean pharmacokinetic profile after
oral administration of 1000 mg VCV tablet
to 41 healthy subjects
Ref: Yadav, 2009
39
MRM example: Improve Sensitivity for
Corticosteroid Detection
• Used illegally as growth promoters in cattle
• Purpose: detect low residue levels in biological matrices
• QQQ mass spectrometer (QuattroLC, Micromass)
• Studied fragmentation of corticosteroids by CID
– Determined negative mode to produce more specific ions
• Evaluated 3 acquisition methods in negative mode
– Product ion
– Neutral loss
– Multiple reaction monitoring
Ref: Antignac, 2000
Improving Sensitivity for Corticosteroid Detection
40
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
Improving Sensitivity for Corticosteroid Detection
MRM = best method
requires setting many transitions
for mixture analysis
Q1 set for multiple [M+acetate]Q3 set for 2 products of each
(-60 and -30 from M+acet]-
41
MRM chromatograms of mixture of 11 steroids
Advantages for selected ion monitoring
Select
Dissociate
Select
NOTES
QQQ
Q-trap
Q-TOF
TOF-TOF
Ion Trap (3D, LT)
ICR
Q or Trap-ICR
LT-Orbitrap
42
MS/MS Scan Modes Summary
Product Ion Scan
Qualitative Structural Information
Select
Dissociate
Scan
Scan
Dissociate
Select
Scan
Dissociate
Scan
Select
Dissociate
Select
Precursor Ion Scan
Screen for compound types
that lose a detectable fragment
Neutral Loss Scan
Screen for compound types
that lose a neutral
Selected Reaction
Monitoring (SRM)
Identify specific compounds
MS/MS Scan Modes Strategy: Phosphorylation
of serine, threonine or tyrosine
16+16+16+31 = 79
phosphorylation
added to serine:
79-1+2 = 80
serine
could be lost from serine
(as an ion):
79 (PO3-)
C
12
H
1
O
16
P
31
could be lost from serine
(as a neutral):
80 + 18 (H2O)= 98
threonine
tyrosine
43
Suggested Reading List & References
Precursor Ion and Neutral Loss Scans
Hopfgartner G., Husser C., Zell M.; Rapid Screening and characterization of drug
metabolites using a new quadrupole-linear ion trap mass spectrometer, JMS,
2003; 38: 138-150.
Triolo A, Altamura, M., Cardinali, F., Sisto, A., Maggi C., Mass spectrometry and
combinatorial chemistry: a short outline, JMS, 2002; 36:1249-1259.
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