Download Tandem Mass Spectrometry: A new diagnostic technology

Mass Spectrometry:
An enabling technology for
biomedical research
New Applications for Mass
Spectrometry Technology
Genomics (Genotype)
•Genetic disease markers (e.g. SNP’s)
Proteomics (Phenotype)
•Protein based disease markers
‘Metabolomics’ (Chemotype)
• Metabolite based disease markers
•The ultimate expression of a disease
These 3 application
areas represent
new and exciting
opportunities for
mass spectrometry.
The 3 areas are
closely related to
one another and to
human health.
Important Recent Developments
in Biological Mass Spectrometry
• API Ionization-MSMS (ITD or QqQ)
– Electrospray
– APCI
• MALDI-TOF
• Qq-TOF
• FT-MS with MALDI and ESI
Fastest Growing Applications
• Biomedical
– Proteomics
– Genomics
– Clinical “Metabolomics” (metabolic disorders, TDM)
• Pharmaceutical
– Preclinical pharmacology (Drug discovery)
– Combinatorial chemistry (Drug discovery)
– Clinical trials (Drug development)
Atmospheric Pressure
Ionization (API)
A
A
A
A+
MS
Before API
e.g. GC/MS
Liquid Introduction Mass Spectrometry
A
A = Analyte;
A
= Solvent;
A+
MS
After API
e.g. LC/MS
= Vacuum system
API Analytical Domains
Ionic
Analyte Polarity
Ion Spray
(Nebulizer Assisted
Electrospray)
Heated Nebulizer
(APCI)
GC/MS
Neutral
101
102
103
Molecular Weight
104
105
IonSpray™ Ion Source
IonSpray = nebulizer assisted electrospray (ESI)
Heated Nebulizer (APCI)
Gas Curtain Interface
Tandem Mass
Spectrometer Analyzers
The API 2000
Triple Quadrupole MSMS
MSMS Ion Optics
Ion
source
Interface
Analyzer
Ion
creation
Ion
Ion
Ion
Ion
transfer
focussing
selection fragmentation
and desolvation
• New Patented LINAC Collision Cell
• New High Efficiency Vacuum System
• Compact Size
• Pulse Counting Detector
Product ion
Ion
selection detection
Mass Analysis
Phe - Molecular Weight= 165.08
9
1
11
2
of C (12.000)
of N (14.003)
of H (1.0080)
of O (15.995)
O
H2N
CH C
CH2
OH
MSMS Ion Optics
Ion
source
Interface
Analyzer
Ion
creation
Ion
Ion
Ion
Ion
transfer
focussing
selection fragmentation
and desolvation
• New Patented LINAC Collision Cell
• New High Efficiency Vacuum System
• Compact Size
• Pulse Counting Detector
Product ion
Ion
selection detection
Quadrupole Mass Filter
Butyl-Ester of Phe (MW= 221.14)
Acquired by NL 102 Exp’t
O
CH
C
OC4H9
100
222.1
2H
-Phe
0
CH 2
2H 13C -Phe
0
1
% Intensity
H2N
2H
-Phe
5
227.1
220
230
m/z, amu
MSMS Ion Optics
Ion
source
Interface
Analyzer
Ion
creation
Ion
Ion
Ion
Ion
transfer
focussing
selection fragmentation
and desolvation
• New Patented LINAC Collision Cell
• New High Efficiency Vacuum System
• Compact Size
• Pulse Counting Detector
Product ion
Ion
selection detection
MSMS of Tryptophan Bu Ester
Scan Types
Ion Path
• Q0 and LINAC Patented for use at high pressures
• Detector floated several kV
• Full Autotune (calibration, resolution and optimization)
Ion Path - Linac
• Q2 rods are tilted and separate DC potentials are
applied to each pair of rods to create an axial
electric field
• Q2 Linac (linear accelerator) eliminates cross-talk
and allows faster MS/MS scanning without
sensitivity losses
• Linac collision cell used at high pressures
demonstrates 100% efficiencies in both product
ion formation and transmission
Ion Path - Linac
Entrance
y
x
x > 2Ro = y
Exit
y
x
V1
V2
• Terminating geometries of the Linac collision cell
LC/MS/MS Scan Modes
Triple Quadrupole Analyzer
•
•
•
•
Precursor Scan (PS)
Product Ion Scan (PI)
Multiple Reaction Mode (MRM)
Neutral Loss Scan (NL)
Product Ion Scan (PI)
Precursor Ions
Q1
Product Ions
*
Q2*
*
* * *
*
Q3
*
**
** * * *
*
* *
* *
CAD GAS
DETECTOR
(CEM)
Product Ion Scans
Illustrated
MS
PQZ
MS/MS
FGC
FG
FGC
HJC
CBA
ABC
WXZ
While Q1 scans,
Q3 detects “C” ions
GC
G
DEC
KLC
F
C
MOLECULAR MASS
MOLECULAR MASS
123
1, 2, 3, 1-2, 2-3
e.g.
Collisional Fragmentation
F, G, C, FG, GC
FGC
Collisional Fragmentation
MSMS of Tryptophan Bu Ester
Multiple Reaction Monitoring
(MRM)
CAD
Precursor Ion
Q1
Q1 scan
for a fixed mass
however more
than one molecule
may have the
same mass
*
Q2*
*
* ** *
*
**
** * * *
*** * *
CAD GAS
Q2 Collision
Product Ion
Q3
DETECTOR
(CEM)
Q3 Scan for another fixed
mass as a component of the
desired molecule
Homocysteine by LC-MSMS
NH2
HOOC
SH
Homocysteine
Exact Mass: 135.04
Mol. Wt.: 135.18
Cardiovascular Risk Factor: mechanism currently
unknown, however, believed will become as important to
cardiovascular health and wellness as cholesterol
Acknowledgements: Dr. Piero Rinaldo, et. al. from Clinical Chemistry, 45(1999)1517
Homocysteine:
MSMS Product Ion Spectrum
[M+H-HCO2H]+
[M+H]+
Homocysteine in Plasma:
LC-MSMS Response (15 mM)
140+
94+
136+
90+
Homocysteine in Plasma:
LC-MSMS Response (0.8 mM)
Homocysteine Method Comparison
LC-MSMS vs. IMx
40.00
y = 0.9797x + 0.2047
LC-MSMS Conc. (mM)
35.00
2
R = 0.9767
30.00
25.00
20.00
15.00
10.00
5.00
0.00
0
5
10
15
20
IMX Conc. (µM)
25
30
35
40
Homocysteine:
HPLC vs. LC-MSMS
• Equipment costs are $0.14 greater per assay
for LC-MSMS vs. HPLC, however…
• 1 LC-MSMS replaces 5 HPLC’s
• Supply costs are 35% less for LC-MSMS
• Space requirements 80% less for LCMSMS
• Personnel costs 29% less for LC-MSMS
• Turnaround time 81% less for LC-MSMS
MSMS experiments for the determination of
Amino acid and Acylcarnitine “Panels”
Amino acids
Acylcarnitines
Neutral loss of 102 & 119 Da
Product Ion Scan of 85 Da
O
R
RC-O
O
NH2-CH-COC4H9
O
O
+
(CH3)3N-CH2-CH-CH2-COC4H9
(CD3 )
H-COC4H9
O
NL 102 Da
+
NH2 = CH-R
+
CH2-CH = CH-COH
PS of 85
NL 102, PS of 85 and MRM Experiments Run Concurrently
Covering Over 50 Analyses (360 channels of information)
Precursor Ion Scan (PS)
OR
CH 3
CH 3 N
CH 3
CH 2 CH
CH 2 C
CAD
+
H 2C
O
Acylcarnitines
Q1
OC 4H 9
*
CHCHCO2H
m/z 85
* *
* * *
* * *
* * * *
* * *
*CAD
* GAS
Q2
Q3
Detector
(CEM)
Control Subject Profile
(MSMS PS 85)
Acylcarnitine Profile from a Normal Control Blood Spot
100
C2 C2
INTERNAL STANDARDS
% Intensity
C16
C8
50
C18:1
C4
C3
C16
C3
C4DC
C5OH
270
300
330
C14:1
360
390
420
m/z, amu
Acknowledgement: Dr. Don Chace
450
480
510
540
Medium Chain Acyl CoA Dehydrogenase
Deficiency (MCAD) (MS/MS PS 85)
Acylcarnitine Profile from a Filter Paper Blood Spot: MCAD
100
1.16 - 25.8 µM
vs. 0.22 µM
C8
% Intensity
INTERNAL STANDARDS
50
C16
C2
C8
C10:1
C2
C4
C6
C10
C16 C18:1
C3
270
300
330
360
390
420
450
Acknowledgement: Dr. Don Chace
480
510
540
Amino acidopathies:
Phenylketonuria (PKU)
O
O
H 2N
CH C
OH
CH2
H 2N
CH C
OH
CH2
CO2, H2O
Phenylalanine
Phenylalanine Hydroxylase
OH
Tyrosine
Neutral Loss Scans (NL)
H
R
O
H
+
H
CAD
N
C
H
H
C
OC
4
H
N
C
9
H
H
-HCO 2C 4 H 9
Amino acids (Bu esters)
Q1
R
+
Butyl formate
(mass 102)
*
*
Q2
*
*
* *
* * *
** * * *
* * * * *
CAD GAS
Q3
DETECTOR
(CEM)
Control Subject Profile
(MSMS NL 102)
Amino Acid Profile from a Normal Control Blood Spot
100
Pro
INTERNAL STANDARDS
% Intensity
Ala
Phe
50
Leu + Ile
Phe
Leu
Gln
Val
Ala
Gly
Gly
140
Ser
Val
160
180
Tyr
Tyr
Cit
Met
200
220
m/z, amu
Glu
Asp Glu
240
260
Acknowledgement: Dr. Don Chace
280
300
Phenylketonuria (PKU)
(MS/MS NL 102)
Amino Acid Profile from a Filter Paper Blood Spot: PKU
Phe
100
Pro
% Intensity
INTERNAL STANDARDS
Ala
50
Phe
Leu + Ile
Gln
Val
Ala
Gly
Gly
140
Ser
160
Val
180
Leu
Cit
Met
200
220
m/z, amu
Tyr Glu
Asp Glu
Tyr
240
Acknowledgement: Dr. Don Chace
260
280
300
Quadrupole Ion Trap
Schematic Representation of Ion Trap Operation
(a) Schematic representation of working points (that is, coordinates in az, qz space) in the stability diagram for
several species of ions stored concurrently. The arrangement of working points with respect to mass/charge ratio is
depicted by figures which differ in size. (b) Ions are shown residing near the bottom of their respective axial
potential wells of depth Dz; the ladder represents the opportunity for resonant ejection of an ion species.
Schematic representation of a quadrupole mass filter and an ion trap,
where f o is the potential applied to opposite pairs of rods or and
caps
az = ax = -ay = 4zU / mw 2ro2
qz = qx = -qy = 2zV / mw 2ro2
Tandem Mass Spectrometry:
Quadrupole Ion Trap
Tandem-in-Time
Eject > M
Daughter
Ion Scanning
Eject < M
CAD
rf
Injection
Ionization
0
Precursor Ion Isolation
Collisionally Activated Dissociation
Product Ion Scanning
Time (ms)
MSn
• since product ions are formed in the same device
in which they are generated, it is possible to
perform multiple stages of MS (MSn)
• genealogical
elucidation
information
facilitates
structure
• for protein/peptide sequencing, low mass B and Y
ions can be generated, thereby negating any
disadvantages due to a low mass cut-off of
1/4*parent mass
Introduction
• Apigenin (m/z 271) is a base component of
flavonoids
• Apigenin has a characteristic fragmentation
spectrum
• MSn of suspected flavonoids can yield the base
component ion m/z 271
• Subsequent fragmentation of the m/z 271 ion can
verify the compound is a flavonoid
ESI-MS
MH+
100
271.2
OH
95
O
90
85
80
75
OH
O
Relative Abundance
70
65
OH
60
Apigenin
55
50
MW 270
45
40
35
30
25
20
15
10
5
186.7 210.7 237.0
0
100
150
200
250
274.0
300
358.4 380.3
350
m/z
422.3
400
479.1
450
524.0 548.5
500
550
600
2
MS
153.1
100
MS/MS of Apigenin
95
90
85
80
75
Relative Abundance
70
65
60
55
50
45
40
35
30
243.3
25
225.3
20
15
145.1
10
5
79.8 90.9
107.3 118.8
121.2 144.1
0
80
100
120
140
271.2
203.2
229.1
163.2
173.0
211.6
197.1
219.4
185.1
160
180
m/z
200
220
253.2
240
260
279.8
280
297.0
300
ESI-MS
579.1
Suspected Flavonoid
100
95
90
85
80
75
Relative Abundance
70
65
60
601.1
55
50
45
40
271.3
35
30
25
602.2
20
15
518.5
10
5
0
200
223.1
263.1 285.3 309.4
250
300
345.1 365.2
350
393.3
419.5
433.2
400
463.4 486.0 516.9 534.8
450
m/z
500
617.5
623.1
645.1
551.5
550
600
650
MS/MS of m/z 579
271.2
100
95
Unknown
90
85
O
OH
80
CH
75
3
Relative Abundance
70
H
65
OH
60
OH
55
50
45
40
35
30
432.9
25
20
(-147)
15
10
5
0
161.6
203.2 225.2
200
270.3 272.2
250
313.2
300
337.1
417.0
366.8 381.0
350
400
m/z
436.5
450
475.0 497.0 518.3 536.5
500
561.7
550
579.3
584.2
600
MS/MS/MS
271.2
100
579>433>products
95
90
(-147)
85
(-162)
80
O
75
Relative Abundance
70
O
O
OH
CH 3
65
OH
60
H
55
OH
Unknown
CH 2
H
OH
OH
OH
50
45
40
35
30
25
20
433.0
15
415.1
10
5
215.2 228.9 270.4 271.8
0
150
200
250
313.3
300
367.1
337.1
397.1
517.2 542.5
448.6
350
m/z
400
450
500
550
600
MS4
153.1
100
95
579>433>271>products
90
85
80
75
Relative Abundance
70
65
60
55
50
45
40
35
30
243.3
25
225.3
20
15
145.1
10
5
79.8 90.9
107.3 118.8
121.2 144.1
0
80
100
120
140
271.2
203.2
229.1
163.2
173.0
211.6
197.1
219.4
185.1
160
180
m/z
200
220
253.2
240
260
279.8
280
297.0
300
MS/MS Apigenin
153.1
100
95
O
OH
90
85
Relative Abundance
80
75
70
65
OH
60
O
55
50
45
OH
40
35
243.3
30
25
225.3
20
145.1
15
10
5
107.3
79.8
90.9
0
80
121.2
102.4
100
163.2
118.9
120
173.0
144.1
140
160
203.1
197.1
176.8
185.1
271.2
229.1
211.6
253.2
279.8
219.4
180
200
220
240
260
m/z
153.1
280
297.0
300
O
OH
100
MS4 m/z 579
95
90
85
Relative Abundance
80
75
O
70
65
CH 2
CH 3
60
H
50
OH
O
OH
55
45
O
O
O
OH
OH
H
OH
OH
OH
40
35
243.3
30
25
225.3
20
145.1
15
10
163.2
5
107.3
79.8
90.9
118.9
121.2
102.4
144.1
0
80
100
120
140
160
176.8
185.1
180
197.1
211.6
253.2
279.8
219.4
200
m/z
271.2
229.1
203.1
173.0
220
240
260
280
297.0
300
Multiply Charged Ions
for Biopolymer Analysis
IonSpray-MS of Diluted Blood
• Full scan mass spectrum of 10 µL blood diluted 500 fold
18+
841.3
100
90
19+
797.1
17+
890.8
80
Ions chosen for 'SIM'
70
16+
946.4
% Intensity
60
ß17+
934.4
50
40
30
ß20+
ß19+
836.1
ß16+
992.7
ß18+
882.5
ß15+
15+
1009.4 1058.8
14+
1081.4
794.4
20
ß14+
1134.3
13+
1164.6
ß13+
1221.5
10
800
900
1000
m/z, amu
1100
1200
12+
1261.5
Hemoglobin MW Spectrum
• Data transformed from m/z to Mr (Da)
100
-hemoglobin
15126.4
ß-hemoglobin
90
15867.1
80
%GHb = 50 {[ g/( +g) + [ßg/(ß+ßg )]}
70
= 50{[8.7/(100+8.7) + [8.7/(82.1+8.7)]}
% Intensity
60
= 8.8%
50
40
30
20
10
Glycated-
Glycated-
16029.2
15288.5
15501.7
15000
15500
16000
Mr (Da)
16500
Hemoglobin Variants
• e.g. sickle cell hemoglobin shows base-line resolution
-hemoglobin
15126.4
90
80
70
% Intensity
60
50
40
ß-hemoglobin
SC-ß-hemoglobin 15867.1
15836.1
30
20
10
Glycated-SC- 
15998.0
Glycated- 
16029.2
Glycated- 
15288.5
15200
15400
15600
15800
16000
Mass, amu
16200
16400
16600
Protein/Peptide Molecular Weights
by Charge State “Deconvolution”
Protein/Peptide
Ubiquitin (bovine)
Cytochrome C (bovine)
Lysozyme (chicken egg)
Hemoglobin-alpha chain (bovine)
Hemoglobin-beta chain (bovine)
Apomyoglobin (equine)
B-lactoglobulin A (bovine milk)
Carbonic Anhydrase (bovine erythrocytes)
Bovine Serum Albumin
Theoretical
Experimental %Mass
Avg. MW
Avg. MW
Accuracy
8564.9
12230.9
14306.2
15053.2
15953.3
16951.5
18363.3
29024.6
66430.3
8565.0
12231.5
14305.0
15053.7
15954.1
16951.1
18364.5
29025.2
66432.3
0.001
0.005
0.008
0.003
0.005
0.002
0.006
0.002
0.003
Average % Mass Accuracy = 0.004%
Schematic of MALDI-TOF
Mirror
Laser
Sample
Tray
Reflectron Flight Tube
Detector
Sample Preparation
for MALDI-TOF Analysis
MALDI-TOF Apparatus
v = (2zVacc / m)1/2; t = (m / 2zVacc)1/2L; t = a (m/z)1/2 + b
TOF Principles
TOF MS
Advantages:
Drawbacks:
Parallel detection of all ions
Originally, low mass resolution
Virtually unlimited m/z range
Requires complicated electronics
for spectra recording:
time-to-digital converter (TDC)
or
transient recorder
No slits or apertures, rods or magnets
High mass accuracy
Limited dynamic range (with TDC)
Typical TOF parameters:
Length ........................................................
Accelerating voltage .................................
Drift time .....................................................
Mass resolution (FWHM) ..........................
20 cm to 5 m
1 to 30 kV
5 to 200 µs
up to 35,000
(Bergmann et al., 1989)
Mass range ................................................. up to ~1 Megadalton
Factors limiting resolution
R= m/Dm=t/2Dt
Dt
Reflecting TOF, or “Reflectron”
MALDI-TOF of Protein Digest:
Adenylate Kinase
17 peptides
MALDI-TOF of Protein Digest:
matching peptides and mass tolerance
MALDI-TOF of Protein Digest:
matching peptides and mass tolerance
MALDI-TOF of Protein Digest:
search results
MALDI-TOF of Protein Digest:
sequence coverage
Short Video
MALDI-TOF of Oligonucleotides
for SNP’s Analysis
Electrostatic
Ion Mirror
ESI-TOF
University of Manitoba
Standing et al., 1993-95
Field-free
drift region
z
y
x
Conducting
Sheath
Object
plane
Deflection
plates
Beam
optics
Detector
Electrospray
Source
Modulator
~ 10 -7 Torr
D
Quadrupole
rods
~10 -5 Torr
~10 -2 Torr
Vacuum Pumps
~2.5 Torr
ESI-TOF:
The advantages of higher resolution
ESI-TOF of Small Molecules:
The advantages of higher resolution
ESI-TOF of Peptides:
Determination of charge state
ESI-TOF of Peptides:
Determination of charge state
ESI-TOF of Peptides:
Determination of molecular weight
ESI-TOF of Peptides&Proteins:
Molecular weight ranges
No mass discrimination
ESI-TOF of Proteins:
Non-covalent Complexs
clusters of catalase HP II
M.W. up to 1.38 MDa
The QSTAR™ Pulsar
Hybrid LC/MS/MS System
• Hybrid MS /MS
(Quadrapole / TOF)
• Most Accurate Hybrid MS/MS
System
• Accurate molecular weight and
sequence information which can
be used for data base searching
• De Novo Peptide Sequencing
• High Sensitivity Post
Translational Modification
Analyses
• Interface with a choice of:
LC or cap.LCNanospray MALDI plate-
QSTAR: Hybrid Quadrupole TOF
770 L/s
250 L/s Modulator
Focusing grid
Accelerator
column
4 anode detector
Sample
Ions
q0
Q1
q2
10 mTorr
2.5 Torr
Curtain
Gas
Conducting
liner
770 L/s
10-2 Torr
Effective Flight
Path = 2.5 m
Field Free
Drift region
7x10-7 Torr
Ion Mirror
(reflector)
QSTAR: Mass Resolution
Peptide Sequencing by MS/MS
N2
CAD Gas
Q0
Q1
MS - Peptide Mass Fingerprint
Q2
Q3
MS/MS - Peptide tag
QSTAR: MS/MS Sensitivity
QSTAR De Novo Peptide
Sequencing with 18O labeling
Shevchenko et al., 1997
De Novo Peptide Sequencing:
two peptides with same m/z
Peptide Sequencing:
K vs Q (∆m=0.036 Da), and F vs Mox (∆m=0.033 Da)
MS/MS on Large Ions:
MW 4587.33 Da, charge 6+
(sample received from BioVisioN, Hannover, Germany)
Product Ions of Metabolite 565 (m/z 565)
565
234 and 235
HO
NH
N
O
O
O
O
Cl
Cl
N
N
O
MH+ 565
Metabolite 565 (m/z)
HO
NH
N
O
O
O
N
N
O
Cl
O
MH+ 565
Cl
HO
NH
-102
N
H
O
+
O
O
H+
O
N
N
O
Cl
N
O
Cl
N
O
m/z 463
m/z 234
463.1130 Accurate
463.1217 Observed (5.6 ppm)
234.1130 Accurate
234.1164 Observed (14.5 ppm)
O
O
O
N
H+
m/z 235
235.1447 Accurate
235.1464 Observed (7.2 ppm)
m/z 234 and 235 at 8,000 resolution
m/z 234.1164 (14.5 ppm)
m/z 235.1464 (7.2 ppm)
BSA: isotopically resolved 47+ charge state
HiResESI @ 7.0 T
HighResMALDI: DNA 20-mer
R50% > 70.000
HiResMALDI 4.7 T: Equine Myoglobin
HiResMALDI 4.7 T: Cytochrome C
R50% > 80.000 @ 4.7 T
Quick Tuning of Instrumental Parameters e.g. for MS/MS
Wizard to select the basic experiment
Tuning of the ‚Arbitrary Waveform Generator‘
to select only a single ion for MS/MS
You will have simple access to all parameters.
ESI spectrum of
Cytochrome C with one
isotopic peak isolated
from the +13 charge state
of the molecular ion
(ULTIMA 7 T)
LINAC
Collision cell
Duty Cycle = 25%
for the heaviest ions
Duty Cycle < 5%
for light ions
TOF
-4kV
Q2
IQ3
Accelerator
IQ2
Slit
Delay
V
Trapping
Extraction region
4-anode detector
Releasing
Duty Cycle >90%
for a pre-selected ion
QSTAR Pulsar: Q2
Pulsing
Q2, collision cell
IQ3/IRP
IRD
IRW
Ions are Stored in Q2, then Pulsed into the TOF
QSTAR Pulsar: Pulsing ON
• TOF Duty Cycle increase:
»from 5% or less (for low mass
ions) to 100% for the ion of
interest
• Sensitivity Increase:
»10-20x at low mass
»3-5x at higher mass (>500 amu)
Can Pulse MS/MS product ions
as well as Precursor Ions
MS/MS Pulsing:
for post-translational
modification (PTM)
identification
and
for enhanced sensitivity of
product ions
Peptide Immonium Ions:
Nanospray infusion of tryptic digest
m/z 86 (Ile/Leu)
m/z 175 (Arg)
m/z 147 (Lys)
m/z 120 (Phe)
Useful for finding
peptides for MS/MS
in complex, dirty
samples (e.g. in-gel
digests)
Fragment ions of m/z=829 (ALILTLVS) normal spectrum....
10000
5000
0
100
200
300
400
500
300
400
500
....and with trapping in Q2
m/z=86
Gain ~ 17
100k
0k
100
200
m/z
Tryptic digest of myoglobin
Precursor ion scan for m/z=86; with no trapping...
20
0
600
700
800
1000
… and with trapping in Q2
Gain ~ 15
500
0
900
600
700
800
m/z
900
1000
Mixture of lipids:
TOF MS vs Precursor Ion scan
Precursor ion scan: parallel detection of two classes of lipids:
phosphatidylcholines and ceramides
Precursor ion scanning for
Phosphorylated Peptides
100 fmol of b-casein tryptic digest – QSTAR Nanospray
*NEGATIVE
ION*
Full TOF MS
Spectrum
Specific
phosphopeptide scan
Phosphopeptide
Pulsar on
x13 gain
LC-MS/MS Applications
with
Precursor Ion Scans
Information Dependent Acquisition
for LC-MS/MS
IDA Principal:
Survey Scan
Filter/Identify Ion(s) of Interest
?
Not necessarily most intense
ion of interest
Identify Ion(s)
MSMS Ion of Interest
Dynamic process repeated throughout the LC analysis
Protein Identification Using Precursor Ion Scans
and Information Dependent Acquisition (IDA)
0.2 pM/mL solution of BSA tryptic digest
LC:
Column type Aquapore Brownlee (AB) 7 micron
size 1 x 100 mm; 60mL/min; 0.5% Formic acid ACN/H2O
10 mL injection
PE 200 Autosampler
MS:
Precursor Ion scan:
Shimadzu LC-10AVP
m/z from 450 to 950 in 2s with step 1,
collision energy 75 eV
Two Dependent Product Ion scans:
1.5s acquisition,
collision energy 45/30 eV
Quad resolution: Low (2-3 amu)
IDA of a BSA Tryptic Digest
LC: C18 column (1x100mm, 7m); 10mL of 0.2pM/mL injected
IDA: 2s precursor scan followed by 2x1.5s product ion scans
T
L
E
N
V
Product Ion Scans from IDA of BSA Tryptic Digest
(1.5s acquisition time for each)
E
D
Y
V
L
F
A
F
L
S
L
L
G
L
V
O
H
Monoacetylmorphine
Metabolism
O
O
Deacetylation
O
O
N
O
N
O
O
O
Diacetylmorphine
Monoacetylmorphine
MWt =327.38
Formula =C19H21NO4
MW =369.42
Formula =C21H23NO5
Deacetylation
O
OH
O
OH
O
H O
O
O
Glucuronidation
O-methylation
OH
O
O
O
N
N
N
H O
H O
H O
Morphine
Codeine
MW=285.35
Formula =C17H19NO3
MW =299.37
Formula =C18H21NO3
Morphine 3-glucuronide
MW =461.47
Formula =C23H27NO9
Glucuronidation
Glucuronidation
O-methylation
H O
O
H O
O
O
O
OH
O
OH
O
N
O
O
O
OH
N
O
O
H O
OH
OH
Morphine 6-glucuronide
MW =461.47
Formula =C23H27NO9
Normorphine
MW =271.32
Formula =C16H17NO3
N
O
OH
Codeine glucuronide
MW =475.50
Formula =C24H29NO9
Fragmentation Pattern of MAM Metabolites
Common fragments selected for precursor ion scanning:
153.1, 155.1, 165.1, 181.1, 183.1, 191.1 and 193.1
(M+H)+
(M+H)+
(M+H+
Identification of Monoacetylmorphine (MAM)
Metabolites Using Precursor Ion Scans and
Information Dependent Acquisition (IDA)
Urine sample from subject exposed to Monoacetylmorphine
LC:
Column type C18(2) Luna Phenomenex 3 micron
size 2 x 100 mm; 200mL/min; 0.5% Formic acid ACN/H2O
10 mL injection
PE 200 Autosampler
MS:
Precursor Ion scan:
Product Ion scan:
Quad resolution:
Shimadzu LC-10AVP
m/z from 200 to 500 in 2s, step 1 amu,
collision energy 45 eV
1s acquisition,
collision energy 35 eV
Low (2-3 amu)
Monoacetylmorphine Metabolites in Urine:
IDA with Multiple Precursor Ion Scan
Morphine (286)
Codeine (300)
Morphine Gluc. (462)
Codeine Gluc. (476)
Monoacetylmorphine (328)
286.0
Monoacetylmorphine Metabolites in Urine:
IDA with TOF MS as a Survey scan
m/z=278
m/z=265
Morphine Gluc. (462)
Morphine (286); Codeine Gluc. (476)
Monoacetylmorphine (328)
Codeine ??? (300)
MALDI QqTOF
Orthogonal MALDI with Collisional Cooling
Advantages
1. Ion source decoupled from analyzer
stable calibration
diversity of target materials
laser fluence, pulse width not critical
no “ghost” peaks of metastable ions
2. Collisional cooling of ions
reduced fragmentation
3. Quasi-continuous ion beam
higher repetition rate
less peak saturation
possibility to use TDC
4. Compatible with tandem MS
control of degree of fragmentation
same two-point calibration and
mass accuracy in MS/MS mode
Drawbacks
1. Lower sensitivity:
reduced duty cycle
losses at TOF entrance
2. Discrimination against
heavy ions in quadrupoles
and at the detector
MALDI QqTOF of a peptide mixture
Single MS mode
Substance P
Resolution ~ 10 000
Accuracy < 10 ppm
Melittin
20000
Fragment of CD4
Insulin
10000
0
1000
2000
File: 007 mixture, Date: 12/5/1998 9:53
Resolution: 1.2 ns, Display bin: 65.0 ns, Starts: 1
3000
m/z
4000
5000
6000
[Courtesy of Alexander Loboda, Werner Ens and
Ken Standing, University of Manitoba]
Mass spectrum of substance P obtained from 70 amol sample
deposited on the probe; DHB matrix; 1200 laser shots in 60 s
[Courtesy of Alexander Loboda, Werner Ens and Ken Standing, University of Manitoba]
Tryptic mass
map of citrate
synthase:
single MS
and
MS/MS of
tryptic fragment
11
[Courtesy of Alexander Loboda, Werner Ens and
Ken Standing, University of Manitoba]
Protein Analysis Scheme
Digested
Sample
MALDI MS
Protein
Database
search
Peptide
List
de novo sequencing
Match
?
Off-line analysis
On-line analysis
Tag search
Tag search
(EST, PTM)
(EST, PTM)
de novo sequencing
Yes
Remove
identified or
predicted
peptides
Daughter
Lists
Yes
Match
?
No
MALDI MS/MS
ESI
MS/MS
on selected
on max
possible
Peaks
peaks
TMS Analyzer Attributes
MS high
res.
NL*
PS*
MRM*
Product
ion
Cost
Ion
collection
efficiency
Duty
cycle
Ion Trap
Yes
No
No
No
Yes
Low
5%
Low*
Triple
Quads
No
Yes
Yes
Yes
Yes
Medium
90+%
High
QSTAR
(QqTOF)
Yes
Yes
Yes
No
Yes
High
50%
High
FT-ICR
Yes
No
No
No
Very
High
Low
Low
*Commonly used scan modes for NBS
Yes
Acknowledgements
•
•
•
•
•
•
•
•
•
•
Patricia Iliusu (PE Biosystems)
Igor Chernushevich (MDS SCIEX)
Bruce Thomson (MDS SCIEX)
Ron Bonner (MDS SCIEX)
Lorne Taylor (Ocada)
Brian Musselman (Perceptive Biosystems)
Larry Haf (Perceptive Biosystems)
Wade Hines (Perceptive Biosystems)
Don Chace (NeoGen Screening)
Werner Sievers (GSG Instruments)