Transcript Document

Introduction to Mass
Spectrometry
Muhammad Asghar Khan
13-Arid-2141
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PRESS RELEASE
Dec. 18, 2013, 12:15 p.m. EST
The Waters ACQUITY QDa Detector
Named a Top Innovation for 2013 Mass Detector for Chromatography
Receives First Annual Analytical Scientist Magazine Innovation Award
MILFORD, Mass., Dec. 18, 2013 /PRNewswire via
COMTEX/ -- Waters Corporation
(wat:NYSE) announced today that its ACQUITY® QDa(TM)
Detector was ranked among the top innovations of 2013 by
The Analytical Scientist magazine
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Mass Spectrometry
An analytical tool used for measuring molecular mass of a sample
Measures mass better than any other technique. Large biomolecules
within accuracy of 0.01%
Gives information about chemical structures of organic compounds and
for peptide or oligonuleotides
Biotechnology: Analysis of proteins, peptides, oligonucleotides
Pharmaceutical: Drug Discovery, pharmacokinetics, drug metabolism
Clinical: Hemoglobin analysis, drug testing
Environmental: Water Quality, food contamination
Geological: Oil composition
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Accurate molecular weight measurements:
Sample confirmation, to determine the purity of a sample, to verify amino acid
substitutions
Reaction monitoring:
To monitor enzyme reactions, chemical modification, protein digestion
Amino acid sequencing:
Sequence confirmation, de novo characterization of peptides, identification of
proteins by database searching with a sequence "tag" from a proteolytic fragments
Protein structure:
Protein folding, protein-ligand complex formation under
physiological conditions, macromolecular structure determination
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How does a mass spectrometer work?
Sample
Ion source:
makes ions
Mass
analyzer:
separates
ions
Mass spectrum:
presents
information
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an early mass spectrometer
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Mass Spectrometer Block Diagram
High Vacuum System
Inlet
GC/HPLC
Flow injection
Sample plate
Ion
source
ESI
MALDI
FAB
EI
CI
Mass
Analyzer
Turbo
molecular
pumps
Detector
Data
System
Electrospray Ionization
Matrix Assisted Laser Desorption Ionisation
Fast Atom Bombardment
Electron Impact
Chemical Ionisation
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Sample dissolved in polar, volatile solvent pumped in stainless steel capillary (75-150mm)
A high voltage of 3 or 4 kV is applied to the tip of the capillary.
the sample dispersed into an aerosol of highly charged droplets aided by a co-axially
introduced nebulising gas. warm flow of nitrogen as the drying gas diminish droplets in size
by solvent evaporation droplets diminish in size by solvent evaporation.
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Electrospray ionization: ESI
Pressure = 1 atm
Inner tube diam. = 100 um
Sample Inlet Nozzle
(Lower Voltage)
Partial
vacuu
m
MH+
N2
Sample in solution
N2 gas
++++
++
++++
++
++++
++
+++
+++
+++
+
++
+++
+
+++
+ +
+ +
+
+
+ +
+
MH2+
MH3+
High voltage applied
to metal sheath (~4 kV)
Charged droplets
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MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate
Laser
hn
MH+
1. Sample is mixed with matrix (X)
and dried on plate.
2. Laser flash ionizes matrix
molecules.
+/- 20 kV
Grid (0 V)
3. Sample molecules (M) are
ionized by proton transfer:
XH+ + M  MH+ + X.
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•Deals well with thermo labile, non-volatile organic
compounds
•Analysis of proteins, peptides, glycoprotein's,
oligosaccharides, and oligonucleotides
•Sample is pre-mixed with a highly absorbing matrix
compound. The matrix transforms the laser energy
into excitation energy
•Bombardment of sample molecules with a laser light
to bring about sample ionization
•Most commercially available have a pulsed nitrogen
laser of wavelength 337 nm
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Electron Impact Ionization (EI)
•Analytes are bombared with high-energy electrons (usually 70eV)
•As a result of collision, an electron is removed from the analytes (M),
generating a molecular ion M+ (radical cation)
M + e-
M+ + 2e-
Chemical Ionization (CI)
Softer ionization technique Less fragmentation  Easier to find molecular ions
Negative chemical ionization (NCI) and Positive Chemical Ionization (PCI).
PCI (simplified): CH4 + e- CH4+ +2eCH4 + CH4+ CH3 + C H5+
NCI:
CH4 + e- CH4-
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Fast Atom Bombardment Mass Spectrometry (FAB-MS)
The material to be analyzed is mixed with a non-volatile chemical
protection environment called a matrix and is bombarded under vacuum
with a high energy (4000 to 10,000 electron volts) beam of atoms. The
atoms are typically from an inert gas such as argon or xenon
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Mass Analyzer
High Vacuum System
Inlet
Ion
source
Mass
Analyzer
Detector
Data
System
Time of flight (TOF)
Quadrupole
Ion Trap
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Mass analyzers separate ions based on their
mass-to-charge ratio (m/z)
• Operate under high vacuum (keeps ions from bumping
into gas molecules)
• After analytes have been ionized they are separated
according to their mass-to-charge ratio (m/z) in a mass
analyzer (mass filter).
•
•
Quadrupoles and ion traps are common mass filters in
GC-MS systems.
•
Time of flight (TOF) mass filter is very much
•
used nowadays in LC-MS systems
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Quadrupole Mass Analyzer
Uses a combination of RF
and DC voltages to operate
as a mass filter.
• Has four parallel metal
rods.
• Lets one mass pass
through at a time.
• Can scan through all
masses or sit at one
fixed mass.
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Quadrupoles have variable ion transmission modes
m2
m4
m1
m4
m3
m2
m1
m3
mass scanning mode
m2
m4
m1
m3
m2
m2
m2
m2
single mass transmission mode
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Time-of-flight (TOF) Mass Analyzer
Source
Drift region (flight tube)
+
+
+
detector
+
V
Separates ion with the same kinetic
energy but different m/z, because
heavier ions require more time to
travel a fixed distance
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QSTARTM ESI QQ TOF or MALDI QQ TOF
Sample
Q0
Q1
Q2
Effective Flight
Path = 2.5 m
Ion Mirror
(reflector)
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Top View
Ion Trap Mass Analyzer
•The ion trap uses three electrodes
to trap ions in small volumes.
•Various voltages are applied to
the ring electrodes as well as to
the entrance and exit endcap
electrodes. A cavity is created
were the ions are trapped.
•Depending on different voltage
settings, ions at a specific m/z is
ejected and detected
Cut away side view
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Detector
High Vacuum System
Inlet
Ion
source
Mass
Analyzer
Detector
Data
System
Microchannel Plate
Electron Multiplier
Hybrid with photomultiplier
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Ions are detected with a microchannel plate
primary ion
- 1000V
+
eee- e
L
- 100V
D
L >> D
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Electron Multiplier EM
The EM multiplies incident charges, thereby amplifying the signal. The
current is measured that is proportional to the amount of analyte in the
sample.
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Data System
High Vacuum System
Inlet
Ion
source
Mass
Analyzer
Detector
Data
System
PC
Sun SPARK Station
DEC Station
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Protein identification by Peptide Mass Fingerprinting (PMF)
Tandem Mass Spectrometer
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Protein Backbone
H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH
N-terminus
Ri-1
AA residuei-1
Ri
AA residuei
Ri+1
C-terminus
AA residuei+1
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Peptide Fragmentation
Collision Induced Dissociation
H+
H...-HN-CH-CO
Ri-1
Prefix Fragment
. . . NH-CH-CO-NH-CH-CO-…OH
Ri
Ri+1
Suffix Fragment
• Peptides tend to fragment along the backbone.
• Fragments can also loose neutral chemical groups
like NH3 and H2O.
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Breaking Protein into Peptides and
Peptides into Fragment Ions
• Proteases, e.g. trypsin, break protein into peptides.
• A Tandem Mass Spectrometer further breaks the
peptides down into fragment ions and measures the
mass of each piece.
• Mass Spectrometer accelerates the fragmented ions;
heavier ions accelerate slower than lighter ones.
• Mass Spectrometer measure mass/charge ratio of an
ion.
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Tools needed
1) Separate
electrophoresis (1D, 2D), Isoelectric focusing
liquid chromatography, affinity, …
B A
d yx z W 1 a2 P n
2) Analyse
mass spectrometry
Mass spectrometer
database correlation
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MS-based protein identification :
concept
Protease
digestion
(Trypsin)
Sequence
database
Software
(MASCOT, SEQUEST,…)
Peptide
extraction
m/z
m/z
MS
Theoretical MS data
m/z
Experimental MS data
Best matching
Sequence(s)
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MALDI-TOF of a tryptic digest of a protein
Extracted peak list
+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ...
a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded
927.59
m/z
…
190
180
170
160
150
140
In te n s ity , c o u n ts
847.5896
869.0722
922.5712
923.5815
927.5904
1022.5551
1050.5533
1163.7695
1164.7531
1193.7393
1249.7705
1250.8103
1296.8556
1297.8499
1305.8668
1416.8929
1440.0008
1479.9773
1482.9583
1567.9417
1640.1635
1824.06
Max. 1305.0 counts.
130
120
110
847.59
100
90
1440.00
80
1479.98 1567.94
70
869.07
60
1640.16
1022.56
50
1163.77
40
1249.77
30
20 789.53
857.14
10
0
871.07
800
900
1024.56
1050.55
978.60
1000
1073.03
1481.98
1296.86
1283.91
1142.86
1108.71
1100
1305.87
1200
1417.93
1386.76 1443.01 1595.95
1824.09
1292.95
1501.84
1790.10
1616.92
1300
1400
m/z, amu
1500
1600
1700
1800
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Matching of MS / MS data
b2
251.12
+TOF Product (653.3)
19.7
19.0
Residue
Immonium a
b
y
----------------------------------------------------------------------------------H, His
110.07
110.07
138.06
1305.71
L, Leu
86.09
223.15
251.15
1168.65
V, Val
72.08
322.22
350.21
1055.57
D, Asp
88.03
437.25
465.24
956.50
E, Glu
102.05
566.29
594.28
841.47
P, Pro
70.06
663.34
691.34
712.43
Q, Gln
101.07
791.40
819.39
615.38
N, Asn
87.05
905.44
933.44
487.32
L, Leu
86.09
1018.53
1046.52
373.28
I, Ile
86.09
1131.6157 1159.61
260.19
K, Lys
101.10
1259.7106 1287.70
147.11
18.0
17.0
16.0
15.0
a1
14.0
I
110.06
13.0
a2
223.13
12.0
Intensity, counts
11.0
10.0
9.0
MH22+
precursor
8.0
b1
7.0
138.05
y1
653.36
6.0
332.21
5.0
b5
I
4.0 86.09
I
3.0
2.0
1.0
594.25
b3
y6
y9
350.24
712.46
a5
1055.49
y3 b4
y7
y8
y10
373.30465.16566.32
166.05
841.53 956.41
1168.56
b8
206.16
Black : predicted
Red : predicted and detected
y2
0.0
100
200
300
400
500
600 700 800
m/z, amu
900 1000 1100 1200 1300
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Thank you
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