Mass Spectrometry 101
An Introductory Lecture On Mass Spectrometry Fundamentals
Presented to the Sandler Mass Spectrometry Users Group University of California San Francisco
April 11, 2003
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 natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers
Applications of Mass Spectrometry
Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical
How does a mass spectrometer work?
Sample
Ion source: makes ions
Mass analyzer: separates ions
Mass spectrum: presents information
Mass Spectrometer Block Diagram
High Vacuum System
Inlet
Ion source
Mass Analyzer
Detector
Data System
Mass Spectrometer Block Diagram
Turbo molecular pumps
High Vacuum System
Inlet
Ion source
Mass Analyzer
Detector
Data System
Sample Introduction
High Vacuum System
Inlet
Ion Source
Mass Analyzer
Detector
Data System
HPLC Flow injection Sample plate
Ion Source
High Vacuum System
Inlet
Ion Source
MALDI ESI FAB LSIMS EI CI
Mass Analyzer
Detector
Data System
Ion Sources make ions from sample molecules
(Ions are easier to detect than neutral molecules.)
Electrospray ionization:
Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage) Partial vacuum
N2 Sample in solution N2 gas
+++ + ++ + + + + ++ + + + + ++ + ++ + ++ + ++ + ++ + ++ + ++
MH+
+ + + + + + + + + +
MH2+
MH3+
High voltage applied to metal sheath (~4 kV)
Charged droplets
MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate hn Laser
MH+
1. Sample is mixed with matrix (X) and dried on plate.
2. Laser flash ionizes matrix molecules.
3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.
+/- 20 kV
Grid (0 V)
Mass Analyzer
High Vacuum System
Inlet
Ion source
Mass Analyzer
Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS
Detector
Data System
Mass analyzers separate ions based on their
mass-to-charge ratio (m/z)
Operate under high vacuum (keeps ions from bumping into gas molecules) Actually measure mass-to-charge ratio of ions (m/z) Key specifications are resolution, mass measurement accuracy, and sensitivity. Several kinds exist: for bioanalysis, quadrupole, time-offlight and ion traps are most used.
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.
Quadrupoles have variable ion transmission modes
m2
m1
m4
m4
m3
m3
m2
m1
mass scanning mode
m2
m1
m4
m3
m2
m2
m2
m2
single mass transmission mode
Time-of-flight (TOF) Mass Analyzer
Source
+
Drift region (flight tube)
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.
detector
Ion Trap Mass Analyzer
Top View
Cut away side view
Detector
High Vacuum System
Inlet
Ion source
Mass Analyzer
Detector
Data System
Microchannel Plate Electron Multiplier Hybrid with photomultiplier
Ions are detected with a microchannel plate
primary ion
- 1000V
+
e-
ee- e
-100V D
L >> D
Data System
High Vacuum System
Inlet
Ion source
Mass Analyzer
Detector
Data System
PC Sun SPARK Station DEC Station
The mass spectrum shows the results
MALDI TOF spectrum of IgG
40000
MH+
Relative Abundance
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
150000
200000
Mass (m/z)
ESI Spectrum of Trypsinogen (MW 23983)
1599.8
M + 15 H+ M + 14 H+
M + 16 H+
1499.9
1714.1
M + 13 H+
1411.9
1845.9 1999.6 2181.6
m/z
Mass-to-charge ratio
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: MALDITOF, ESI-Quad.
-Analyzers can be combined to create hybrid instruments. ESI-QQQ, MALDI QQ TOF, Q Trap
Voyager-DE STR MALDI TOF
Sample plate Extraction grids Laser Linear detector
Reflector Timed ion detector selector Reflector
Camera Pumping Pumping
QSTARTM ESI QQ TOF or MALDI QQ TOF
Sample
Q0 Q1 Q2
Effective Flight Path = 2.5 m
Ion Mirror (reflector)
QTRAP: Linear Ion Trap on a Triple Quadrupole A new type of instrument.
Q0
Q1
Q2
Q3
Exit
linear ion trap
Summary: acquiring a mass spectrum
Ionization Ion Source
Form ions (charged molecules)
Mass Sorting (filtering)
Detection Ion Detector
Detect ions
Mass Analyzer
Sort Ions by Mass (m/z)
100 75
Inlet
Solid Liquid Vapor
50 25 0 1330 1340 1350
Mass Spectrum
How is mass defined?
Assigning numerical value to the intrinsic property of mass is based on using carbon-12, 12C, as a reference point. One unit of mass is defined as a Dalton (Da). One Dalton is defined as 1/12 the mass of a single carbon-12 atom. Thus, one 12C atom has a mass of 12.0000 Da.
Isotopes
+Most elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 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 these isotopes, better mass accuracy is achieved.
Stable isotopes of most abundant elements of peptides
Element H C N O Mass 1.0078 2.0141 12.0000 13.0034 14.0031 15.0001 15.9949 16.9991 17.9992 Abundance 99.985% 0.015 98.89 1.11 99.64 0.36 99.76 0.04 0.20
Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)
Monoisotopic mass
No 13C atoms (all 12C)
1981.84 1982.84 One 13C atom
1983.84 Two 13C atoms
Isotope pattern for a larger peptide (207 C-atoms)
4361.45
4360.45
m/z
Mass spectrum of insulin
2 x 13C
13C
12C :
5730.61
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.
Monoisotopic mass
Monoisotopic mass corresponds to lowest mass peak
When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.
Average mass
Average mass corresponds to the centroid of the unresolved 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.
What if the resolution is not so good?
At lower resolution, the mass measured is the average mass.
Better resolution
Poorer resolution
6130
6140
6150
6160
6170
Mass
How is mass resolution calculated?
100
ISO:CH3
M 15.0229
100
90
80
70
R = M/DM
FWHM = DM
60
% Intensity
50
40
30
20
10
0 15.01500
15.01820
15.02140 Mass (m /z)
15.02460
15.02780
15.03100
Mass measurement accuracy depends on resolution High resolution means better mass accuracy
8000
Resolution =18100 15 ppm error
6000
Counts
24 ppm error
4000
Resolution = 14200
Resolution = 4500
2000
55 ppm error
0 2840 2845 2850 2855
Mass (m/z)
How do we achieve superior mass resolution?
Reflector TOF Mass Analyzer Delayed Extraction on a MALDI source
Important performance factors
Mass accuracy: How accurate is the mass measurement?
Resolution: How well separated are the peaks from each other? Sensitivity: How small an amount can be analyzed?
What is MSMS?
MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information. Mixture of ions Ion source MS-1 Single ion MS-2 Fragments
What is MS/MS?
Peptide mixture
1 peptide selected for MS/MS
+ MS/MS + + + +
Have only masses to start
The masses of all the pieces give an MS/MS spectrum
Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle
+ + + +
Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together
Cleavages Observed in MS/MS of Peptides
yn-i
low energy
zn-i wn-i
xn-i
vn-i
-HN--CH--CO--NH--CH--CO--NH-
Ri
ai
bi ci
CH-R
R
di+1
high energy
Peptide Fragmentation
=>
E=Glu G=Gly S=Ser F=Phe N=Asn P=Pro V=Val A=Ala R=Arg
175.10 246.14 345.21 459.25 556.30 670.35 799.39 928.43 985.45 1132.52 1279.59 1366.62 1423.64 1552.69
= =
Protein Identification
1. Peptide Mass Finger Printing (PMF) from MS data 2. Database search using fragment ion masses from MS/MS data 3. Sequence Tags from MS/MS data
PROBLEM
Bank President
Who robbed the bank?
Biologist
What protein was isolated?
GATHER EVIDENCE
Police Officer
1. Interview witnesses 2. Dust for fingerprints
Mass Spectrometrist
1. Interview biologist who isolated the protein 2. Cleave protein to obtain peptide mixture
enzyme
3. Analyze peptide mixture by MS to obtain peptide molecular masses!
DATABASE SEARCH
Police Officer
Height: 57 Weight: 160 lbs Gender: male Age: 35-40 Fingerprints
Mass Spectrometrist
Approx. molecular weight: 30,000 Origin: bovine liver Peptide mass list from MS analysis: 975.4832, 1112.5368, 632.3147, 803.4134, 764.3892
search
search
PEPTIDE MASS DATABASE OF KNOWN PROTEINS
DATABASE OF KNOWN FELONS
DATABASE SEARCH RESULTS
Police Officer Identifies the robber Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase
Anthony J. Felon
Voyager Spec #1 MC=>AdvBC(32,0.5,0.1)=>NR(1.50)[BP = 1025.5, 26876]
1 02 5.50
Peptide mass fingerprint of Spot A
2.7E+4
100 90 80 70 60 50 40 30 20 10 0 886.0
Gel coordinates: 16kDa, 4.2 (mwt, pI)
% In ten sity
1 34 1.63
1 17 9.60
1 27 7.71
1 23 4.65
9 95 .5 8
1 30 8.66
1 54 4.69
1 78 6.82
1 10 7.56
1 70 8.75
1 99 4.99
2 21 1.10
1165.6
1445.2 Mass (m/z)
1724.8
2004.4
0 2284.0
Mass accuracy tolerance = 15 ppm This means that the mass is within 0.015 Da at m/z 1000
MS/MS spectrum for tryptic peptide MH+ = 1025.5, EAFQLFDR, from Spot A. An MS-Tag search using the fragment ions from this spectrum confirmed the identity of Spot A as myosin light chain.
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
100 90 80 70 60 50 40 30 20 10 0
y 1(+1)
5.1E+4
b2(+1)
b3 - 18(+1),AFQ (+1) - 17
FQ L(+1),Q LF(+1)
b1 - 18(+1)
Q L(+1) - 28
y 1 - 17(+1)
y 3 - 17(+1)
b4 - 18(+1)
165.1
y 2(+1)
Q L(+1)
347.1
365.1
FD(+1)
229.2
84.0
y 3(+1)
70.0
b4(+1),Q LFD(+1) - 28
% In ten sity
a2(+1)
FQ (+1)
65
152
239 Mass (m/z)
326
413
0 500
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
y 4(+1) b5(+1) AFQ LFD(+1) - 17,AFQ LFD(+1) - 18
100 90 80 70 60 50 40 30 20 10 0 500
6735.5
y 8(+1) 714.8 730.1 y 7(+1) 961.0 819.9 b7(+1) 941.5 973.7
% In ten sity
y 4 - 17(+1)
a5(+1)
647.4
610
720 Mass (m/z)
830
940
0 1050
Sequence Tags from Peptide Fragmentation by MS/MS
One sequence tag includes four components:
peptide molecular weight (MW) partial sequence (region 2) molecular wt before partial sequence (region 1) molecular wts after partial sequence (region 3)
Peptide measured molecular wt = 1927.2
A V I/L T
381.1
Partial Sequence - A-V-I/L-T-
1108.13
1546.11
region 1
region 2
region 3
Sequence TAG Example from MS/MS Spectrum Peptide MW = 1345.70
Sequence Tag (739.34)SVS(I/L)(1120.60)
800 y7
700
600
500
y3 b 5 - 2 ( H 2 O)
[M+2H]2+
400
300
I/ L b 4 - H2 O y6 y9
200
739.34
b 5 - H2 O
2 9 4 .2 b 2 - H2 O y 2 b 3 - H2 O a2 b1 b2 b3 200 400 y1
Vy8 S
I/L
y10
100
b4
y 4 b 6 - H2 O y5 b5 600 m/z, amu 800 1000
1120.60
y11 1200
Sequence Tag search identifies 1 hit (carbonic anhydrase)
Acknowledgements
We thank the Applied Biosystems Mass Spectrometry Applications Laboratory for allowing the use of some of their slides for this presentation.