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
Mass Spectrometry 101 An Introductory Lecture On Mass Spectrometry Fundamentals Presented to the Sandler Mass Spectrometry Users’ Group University of California.
Dec 22, 2015
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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 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
Pharmaceutical analysisBioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysisScreening of drug candidatesIdentifying drug targets
Biomolecule characterizationProteins and peptidesOligonucleotides
Environmental analysisPesticides on foodsSoil and groundwater contamination
Forensic analysis/clinical
Applications of Mass Spectrometry
How does a mass spectrometer work?
Ion source:makes ions
Mass analyzer: separates ions
Mass spectrum:presents
information
Sample
InletIonsource
Mass Analyzer Detector
DataSystem
High Vacuum System
Mass Spectrometer Block Diagram
InletIonsource
Mass Analyzer Detector
DataSystem
High Vacuum System
Mass Spectrometer Block Diagram
Turbo molecular pumps
Inlet IonSource
Mass Analyzer Detector
DataSystem
High Vacuum System
HPLCFlow injectionSample plate
Sample Introduction
Inlet IonSource
Mass Analyzer Detector
DataSystem
High Vacuum System
MALDIESIFABLSIMSEICI
Ion Source
High voltage applied to 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.)
h Laser
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.
MH+
MALDI: Matrix Assisted Laser Desorption Ionization
+/- 20 kV Grid (0 V)
Sample plate
InletIonsource
Mass Analyzer Detector
DataSystem
High Vacuum System
Time of flight (TOF)QuadrupoleIon TrapMagnetic SectorFTMS
Mass Analyzer
¤ 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-of-flight and ion traps are most used.
Mass analyzers separate ions based on their
mass-to-charge ratio (m/z)
Quadrupole Mass AnalyzerUses 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.
mass scanning mode
m1m3m4 m2
m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2m3
m1
m4
m2
Quadrupoles have variable ion transmission modes
Time-of-flight (TOF) Mass Analyzer
+
+
+
+
Source Drift region (flight tube)
dete
ctor
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.
Ion Trap Mass Analyzer
Top View
Cut away side view
InletIonsource
Mass Analyzer Detector
DataSystem
High Vacuum System
Microchannel PlateElectron MultiplierHybrid with photomultiplier
Detector
+e-
primary ion
e-
e- e-L
D
-1000V
-100V
L >> D
Ions are detected with a microchannel plate
InletIonsource
Mass Analyzer Detector
DataSystem
High Vacuum System
PCSun SPARK StationDEC Station
Data System
The mass spectrum shows the results
Re
lativ
e A
bun
dan
ce
Mass (m/z)
0
10000
20000
30000
40000
50000 100000 150000 200000
MH+
(M+2H)2+
(M+3H)3+
MALDI TOF spectrum of IgG
ESI Spectrum of Trypsinogen (MW 23983)
1599.8
1499.9
1714.1
1845.91411.9
1999.6
2181.6
M + 15 H+
M + 13 H+
M + 14 H+M + 16 H+
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: “MALDI-TOF,” “ESI-Quad.”
-Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap
Voyager-DE STR MALDI TOF
Camera
Laser
Sample plate
Pumping Pumping
Timed ion selector Reflector
Linear detector
Extractiongrids
Reflectordetector
QSTARQSTARTMTM ESI QQ TOF or MALDI QQ TOF ESI QQ TOF or MALDI QQ TOF
Q1
Ion Mirror(reflector)
Effective FlightPath = 2.5 m
Q2
Q0
Sample
QTRAP: Linear Ion Trap on a Triple QuadrupoleQTRAP: Linear Ion Trap on a Triple Quadrupole
A new type of instrument….
linear ion trap
Exit
Q0 Q1 Q2 Q3
Inlet
Ionization
Mass Analyzer
Mass Sorting (filtering)
Ion Detector
Detection
Ion Source
• 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
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.
How is mass defined?
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.
Element Mass AbundanceH 1.0078
2.014199.985%0.015
C 12.000013.0034
98.891.11
N 14.003115.0001
99.640.36
O 15.994916.999117.9992
99.760.040.20
Stable isotopes of most abundant elements of peptides
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”
m/z
4360.45
4361.45
Isotope pattern for a larger peptide (207 C-atoms)
Mass spectrum of insulin
12C : 5730.61
13C
2 x 13C
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 masscorresponds tolowest 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.
6130 6140 6150 6160 6170
Poorer resolution
Better resolution
What if the resolution is not so good?
At lower resolution, the mass measured is the average mass.
Mass
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
% Int
ensit
yISO:CH3
15.0229M
FWHM = M
R = M/M
How is mass resolution calculated?
Mass measurement accuracy depends on resolution
0
2000
4000
6000
8000
Co
un
ts
2840 2845 2850 2855
Mass (m/z)
Resolution = 14200
Resolution = 4500
Resolution =18100 15 ppm error
24 ppm error
55 ppm error
High resolution means better mass accuracy
How do we achieve superior mass resolution?
Delayed Extraction on a MALDI source
Reflector TOF Mass Analyzer
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.
Ion source
MS-2MS-1
Mixture of ions
Single ion
Fragments
What is MS/MS?
MS/MS+
+
+ +
+
1 peptide selected for
MS/MS
The masses of all the pieces give an MS/MS spectrum
Peptidemixture
Have only masses to start
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
-HN--CH--CO--NH--CH--CO--NH-
Ri CH-R’
ci
zn-i
R”
di+1
vn-i wn-i
low energy
high energy
Cleavages Observed in MS/MS of Peptides
ai
xn-i
bi
yn-i
E G S F F G E E N P N V A R
Peptide Fragmentation
175.10246.14345.21459.25556.30670.35799.39928.43985.45
1132.521279.591366.621423.641552.69
=>
=
=
=
E=GluG=GlyS=SerF=PheN=AsnP=ProV=ValA=AlaR=Arg
Protein Identification
1. Peptide Mass Finger Printing (PMF)from MS data
2. Database search using fragment ion masses from MS/MS data
3. Sequence Tagsfrom MS/MS data
PROBLEM
Bank President
Who robbed the bank?
Biologist
What protein was isolated?
Mass Spectrometrist
1. Interview biologist who isolated 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
DATABASE SEARCH
Police OfficerHeight: 5’7”
Weight: 160 lbs
Gender: male
Age: 35-40
Fingerprints
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
KNOWN FELONS
PEPTIDE MASS DATABASEOF KNOWN PROTEINS
search search
DATABASE SEARCH RESULTS
Police Officer
Identifies the robber
Anthony J. Felon
Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase
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
% Int
ensit
yVoyager Spec #1 MC=>AdvBC(32,0.5,0.1)=>NR(1.50)[BP = 1025.5, 26876]
1025.5
0
1341.6
3
1786.8
2
1277.7
1
1179.6
0
1544.6
9
995.58
1234.6
5
1308.6
6
2211.1
0
1708.7
5
1107.5
6
1994.9
9
Peptide mass fingerprint of Spot A
Gel coordinates: 16kDa, 4.2 (mwt, pI)
Mass accuracy tolerance = 15 ppm
This means that the mass is within 0.015 Da at
m/z 1000
500 610 720 830 940 1050
Mass (m/z)
0
6735.5
0102030405060708090
100
% In
tens
ity
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)
AFQL
FD(+
1) -
17,A
FQLF
D(+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
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]F
y1(+
1)
b2(+
1)
FQ(+
1)
Q
a2(+
1)
b4(+
1),Q
LFD(
+1) -
28
L b1 -
18(+
1)
165.1
365.1
347.1
y2(+
1)
y3 -
17(+
1)
QL(+
1) -
28
y1 -
17(+
1)
70.0
y3(+
1)
84.0
QL(+
1)
b3 -
18(+
1),A
FQ(+
1) -
17
229.2
FQL(
+1),Q
LF(+
1)
FD(+
1)
b4 -
18(+
1)
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.
Sequence Tags from Peptide Fragmentation 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 Peptide MW = 1345.70
y 11
y 10
y 9
y 8
y 7
y 6
y 5y 4
y 3
y 2
y 1
b1 b2 b3 b4 b5
I/ L
a2
294 .2b2 - H2 O
b3 - H2 O
b4 - H2 O
b5 - H2 O
b6 - H2 O
b5 - 2(H2 O)
200 400 600 800 1000 1200m/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 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.
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