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Mass Spectrometry 101
An Introductory Lecture On Mass Spectrometry
Fundamentals
Presented to the Sandler Mass Spectrometry Users Group
University of California San FranciscoApril 11, 2003
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What does a mass spectrometer do?
1. It measures mass better than any other technique.2. It can give information about chemical structures.
What are mass measurements good for?
To identify, verify, and quantitate: metabolites,recombinant proteins, proteins isolated from naturalsources, oligonucleotides, drug candidates, peptides,
synthetic organic chemicals, polymers
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Pharmaceutical analysis
Bioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysis
Screening of drug candidatesIdentifying drug targetsBiomolecule characterization
Proteins and peptidesOligonucleotides
Environmental analysisPesticides on foodsSoil and groundwater contamination
Forensic analysis/clinical
Applications of Mass Spectrometry
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How does a mass spectrometer work?
Ion source:makes ions
Massanalyzer:separates
ions
Mass spectrum:presents
information
Sample
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InletIon
source MassAnalyzer Detector DataSystem
High Vacuum System
Mass Spectrometer Block Diagram
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InletIon
source MassAnalyzer Detector DataSystem
High Vacuum System
Mass Spectrometer Block Diagram
Turbo
molecular
pumps
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InletIon
Source MassAnalyzer Detector DataSystem
High Vacuum System
HPLCFlow injection
Sample plate
Sample Introduction
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InletIon
Source MassAnalyzer Detector DataSystem
High Vacuum System
MALDIESI
FAB
LSIMS
EI
CI
Ion Source
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High voltage appliedto metal sheath (~4 kV)
Sample Inlet Nozzle(Lower Voltage)
Charged droplets
++
+++
+
++
+ +++
++
+ +++
+++
+++
+++
+++
+
+
++
+
+
+
+++
+++
+++
MH+
MH3+
MH2+
Pressure = 1 atmInner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partialvacuum
Electrospray ionization:
Ion Sources make ions from sample molecules(Ions are easier to detect than neutral molecules.)
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hn Laser
1. Sample is mixed with matrix (X)and dried on plate.
2. Laser flash ionizes matrixmolecules.
3. Sample molecules (M) are
ionized by proton transfer:XH+ + M MH+ + X.
MH+
MALDI: Matrix Assisted Laser Desorption Ionization
+/- 20 kV Grid (0 V)
Sample plate
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InletIon
source MassAnalyzer Detector DataSystem
High Vacuum System
Time of flight (TOF)Quadrupole
Ion Trap
Magnetic Sector
FTMS
Mass Analyzer
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Operate under high vacuum (keeps ions from bumpinginto gas molecules)
Actually measure mass-to-charge ratio of ions (m/z)
Key specifications are resolution, mass measurementaccuracy, and sensitivity.
Several kinds exist: for bioanalysis, quadrupole,time-of-
flight and ion traps are most used.
Mass analyzers separate ions based on their
mass-to-charge ratio (m/z)
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Quadrupole Mass Analyzer
Uses a combination of RF
and DC voltages to operateas a mass filter.
Has four parallel metalrods.
Lets one mass passthrough at a time.
Can scan through all
masses or sit at onefixed mass.
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mass scanning mode
m1m3m4 m2m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2
m3
m1
m4
m2
Quadrupoles have variable ion transmission modes
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Time-of-flight (TOF) Mass Analyzer
+
+
+
+
Source Drift region (flight tube)
detector
V
Ions are formed in pulses.
The drift region is field free.
Measures the time for ions to reach the detector.
Small ions reach the detector before large ones.
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Ion Trap Mass Analyzer
Top View
Cut away side view
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InletIon
source MassAnalyzer Detector DataSystem
High Vacuum System
Microchannel PlateElectron Multiplier
Hybrid with photomultiplier
Detector
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+e-
primary ion
e-
e- e-
L
D
-1000V
-100V
L >> D
Ions are detected with a microchannel plate
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InletIon
source MassAnalyzer Detector DataSystem
High Vacuum System
PC
Sun SPARK Station
DEC Station
Data System
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The mass spectrum shows the results
RelativeA
bundance
Mass (m/z)
0
10000
20000
30000
40000
50000 100000 150000 200000
MH+
(M+2H)2+
(M+3H)3+
MALDI TOF spectrum of IgG
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ESI Spectrum of Trypsinogen (MW 23983)
1599.8
1499.9
1714.1
1845.91411.9
1999.62181.6
M + 15 H+
M + 13 H+
M + 14 H+M + 16 H+
m/z Mass-to-charge ratio
H d t t t th i ?
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How do mass spectrometers get their names?
Types of ion sources:
Electrospray (ESI) Matrix Assisted Laser Desorption Ionization (MALDI)
Types of mass analyzers:
Quadrupole (Quad, Q)
Ion Trap
Time-of-Flight (TOF)
-Either source type can work with either analyzer type: MALDI-TOF, ESI-Quad.
-Analyzers can be combined to create hybrid instruments.
ESI-QQQ, MALDI QQ TOF, Q Trap
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Voyager-DE STR MALDI TOF
Camera
Laser
Sampleplate
Pumping Pumping
Timed ionselector Reflector
Lineardetector
Extractiongrids
Reflectordetector
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QSTARTM ESI QQ TOF or MALDI QQ TOF
Q1
Ion Mirror
(reflector)
Effective Flight
Path = 2.5 m
Q2
Q0
Sample
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QTRAP: Linear Ion Trap on a Triple Quadrupole
A new type of instrument.
linear ion trap
Exit
Q0 Q1 Q2 Q3
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Inlet
Ionization
Mass Analyzer
Mass Sorting (filtering)
IonDetector
Detection
IonSource
Solid Liquid Vapor
Detect ionsForm ions
(charged molecules) Sort Ions by Mass (m/z)
1330 1340 1350
100
75
50
25
0
Mass Spectrum
Summary: acquiring a mass spectrum
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Assigning numerical value to the intrinsic propertyof mass is based on using carbon-12, 12C, as areference point.
One unit of mass is defined as a Dalton (Da).
One Dalton is defined as 1/12 the mass of a singlecarbon-12 atom.
Thus, one 12C atom has a mass of 12.0000 Da.
How is mass defined?
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Isotopes
+Most elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but innature, 1.1% of C atoms have an extra neutron, making their mass13 Da.
+Why do we care?
Mass spectrometers can see isotope peaks if their resolution is
high enough.
If an MS instrument has resolution high enough to resolve theseisotopes, better mass accuracy is achieved.
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Element Mass Abundance
H 1.0078
2.0141
99.985%
0.015
C 12.0000
13.0034
98.89
1.11N 14.0031
15.0001
99.64
0.36
O 15.9949
16.9991
17.9992
99.76
0.04
0.20
Stable isotopes of most abundant elements ofpeptides
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1981.84
1982.84
1983.84
Mass spectrum of peptide with 94 C-atoms(19 amino acid residues)
No 13C atoms (all 12C)
One 13C atom
Two 13C atoms
Monoisotopic mass
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m/z
4360.45
4361.45
Isotope pattern for a larger peptide (207 C-atoms)
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Mass spectrum of insulin
12C: 5730.61
13C
2 x13
C
Insulin has 257 C-atoms. Above this mass, the monoisotopic
peak is too small to be very useful, and the average mass is
usually used.
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Monoisotopic mass
Monoisotopic masscorresponds tolowest mass peak
When the isotopes are clearly resolved the monoisotopic mass
is used as it is the most accurate measurement.
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Average mass
Average mass corresponds
to the centroid of theunresolved peak cluster
When the isotopes are not resolved, the centroid of the envelope
corresponds to the weighted average of all the the isotope peaks in
the cluster, which is the same as the average or chemical mass.
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6130 6140 6150 6160 6170
Poorerresolution
Betterresolution
What if the resolution is not so good?
At lower resolution, the mass measured is the average mass.
Mass
ISO:CH3H i l ti l l t d?
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15.01500 15.01820 15.02140 15.02460 15.02780 15.03100
Mass (m /z)
100
0
10
20
30
40
50
60
70
80
90
100
%
Intensity
15.0229M
FWHM = DM
R = M/DM
How is mass resolution calculated?
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Mass measurement accuracy depends on resolution
0
2000
4000
6000
8000
Counts
2840 2845 2850 2855
Mass (m/z)
Resolution = 14200
Resolution = 4500
Resolution =1810015 ppm error
24 ppm error
55 ppm error
High resolution means better mass accuracy
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How do we achieve superior massresolution?
Delayed Extraction on a MALDI source
Reflector TOF Mass Analyzer
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Important performance factors
Mass accuracy: How accurate is the massmeasurement?
Resolution: How well separated are the peaksfrom each other?
Sensitivity: How small an amount can beanalyzed?
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What is MSMS?
MS/MS means using two mass analyzers (combinedin one instrument) to select an analyte (ion) from amixture, then generate fragments from it to givestructural information.
Ionsource MS-2MS-1
Mixture ofions
Singleion
Fragments
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What is MS/MS?
MS/MS+
+
++
+
1 peptideselected for
MS/MS
The masses of allthe pieces give anMS/MS spectrum
Peptide
mixture
Have only massesto start
Interpretation of an MSMS spectrum to derive
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Interpretation of an MSMS spectrum to derivestructural information is analogous to solving
a puzzle
+
+
++
+
Use the fragment ion masses as specific pieces ofthe puzzle to help piece the intact molecule backtogether
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-HN--CH--CO--NH--CH--CO--NH-Ri CH-R
ci
zn-i
R
di+1
vn-i wn-ilow energy
high energy
Cleavages Observed in MS/MS of Peptides
ai
xn-i
bi
yn-i
E Glu
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E G S F F G E E N P N V A R
Peptide Fragmentation
175.10246.14345.21459.25556.30670.35799.39
928.43985.451132.521279.591366.621423.641552.69
=>
=
=
=
E=GluG=GlyS=SerF=PheN=Asn
P=ProV=ValA=AlaR=Arg
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Protein Identification
1. Peptide Mass Finger Printing (PMF)from MS data
2. Database search using fragment ion massesfrom MS/MS data
3. Sequence Tagsfrom MS/MS data
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PROBLEM
Bank President
Who robbed the bank?
Biologist
What protein was
isolated?
GATHER EVIDENCE
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Mass Spectrometrist
1. Interview biologist whoisolated the protein
2. Cleave protein to obtain
peptide mixture
3. Analyze peptide mixture by
MS to obtain peptide
molecular masses!
GATHER EVIDENCE
Police Officer
1. Interview witnesses
2. Dust for fingerprints
enzyme
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DATABASE SEARCH
Police OfficerHeight: 57
Weight: 160 lbs
Gender: male
Age: 35-40Fingerprints
Mass SpectrometristApprox. molecular weight: 30,000
Origin: bovine liver
Peptide mass list from MS analysis:975.4832, 1112.5368, 632.3147,803.4134, 764.3892
DATABASE OF
KNOWNFELONS
PEPTIDE MASSDATABASE
OF KNOWNPROTEINS
search search
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DATABASE SEARCH RESULTS
Police Officer
Identifies the robber
Anthony J. Felon
Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase
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886.0 1165.6 1445.2 1724.8 2004.4 2284.0
Mass m/z
0
2.7E+4
0
10
20
30
40
50
60
70
80
90
100
%I
ntensity
Voyager Spec #1 MC=>AdvBC (32,0.5,0.1)=>NR (1.50)[B P = 1025.5, 26876]
1025.
50
1341.
63
1786.
82
1277.
71
1179.
60
1544.
69
995.
58
1234.
65
1308.
66
2211.
10
1708.
75
1107.
56
1994.
99
Peptide mass fingerprint of Spot A
Gel coordinates: 16kDa, 4.2 (mwt, pI)
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Mass accuracytolerance = 15 ppm
This means that themass is within
0.015 Da atm/z 1000
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MS/MS t f t ti tid MH+ 1025 5 EAFQLFDR f
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500 610 720 830 940 1050
Mass m/z
0
6735.5
0102030405060708090
100
%I
ntensity
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
y4(+1)
b5(+1)
y8(+1)
y4-
17(+1)
a5(+1)
647.4
714.8
y7(+1)
AFQLFD(+1)-
17,A
FQLFD(+1
)-
18
730.1
973.7
961.0
819.9
941.5
b7(+1)
65 152 239 326 413 500
Mass m/z
0
5.1E+4
0
1020
30
40
50
60
70
80
90
100
%I
ntensity
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
F
y1(+1)
b2(+1)
FQ(+1)
Qa2(+1)
b4(+1),QLFD(+1)-
28
L b1-
18(+1)
165.1
36
5.1
34
7.1
y2
(+1)
y3
-17(+1)
QL
(+1)-
28
y1
-17(+1)
70.
0
y3(+
1)
84.0
QL(+1)
b3-
18(+1),AFQ(+1)-
17
229.2
FQL(+1),QLF(+1)
FD(+1
)
b4-1
8(+1)
MS/MS spectrum for tryptic peptide MH+ = 1025.5, EAFQLFDR, fromSpot A. An MS-Tag search using the fragment ions from this spectrumconfirmed the identity of Spot A as myosin light chain.
Sequence Tags from Peptide
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Sequence Tags from PeptideFragmentation by MS/MS
peptide molecular weight (MW)
partial sequence (region 2)
molecular wt before partial sequence (region 1)
molecular wts after partial sequence (region 3)
A V I/L T
Peptide measured molecular wt = 1927.2
1108.13Partial Sequence- A-V-I/L-T-
381.1
region 1 region 2 region 3
One sequence tag includes four components:
1546.11
Sequence TAG Example from MS/MS Spectrum
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Sequence TAG Example from MS/MS SpectrumPeptide MW = 1345.70
y11
y10
y9
y8
y7
y
6
y5
y4
y3
y2
y1
b1 b2 b3b4 b5
I/ L
a2
294.2b2-H2O
b3-H2O
b4-H2O
b5-H2O
b6-H2O
b5-2(H2O)
200 400 600 800 1000 1200
m/z, amu
100
200
300
400
500
600
700
800
Sequence Tag (739.34)SVS(I/L)(1120.60)
739.34
1120.60
[M+2H]2+
S V I/LS
Sequence Tag search identifies
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Sequence Tag search identifies1 hit (carbonic anhydrase)
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Acknowledgements
We thank the Applied Biosystems Mass Spectrometry ApplicationsLaboratory for allowing the use of some of their slides for thispresentation.