Transcript Slide 1 - UCSF Macromolecular Structure Group

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
High Vacuum System
Inlet
Ion
source
Mass
Analyzer
Turbo
molecular
pumps
Detector
Data
System
Sample Introduction
High Vacuum System
Inlet
HPLC
Flow injection
Sample plate
Ion
Source
Mass
Analyzer
Detector
Data
System
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
Partial
vacuum
Sample Inlet Nozzle
(Lower Voltage)
MH+
N2
Sample in solution
N2 gas
+
+ ++
++
++++
++
+
+ ++
++
+
++
+
++
+
+
++
+
++
+
++
+
++
+ +
+
+
+
+
+
+
MH2+
+
MH3+
High voltage applied
to metal sheath (~4 kV)
Charged droplets
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.
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
m4
m1
m4
m3
m2
m1
m3
mass scanning mode
m2
m4
m1
m3
m2
m2
m2
single mass transmission mode
m2
Time-of-flight (TOF) Mass Analyzer
Source
Drift region (flight tube)
+
+
+
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.
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
+
ee-
L
e- 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
MH+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
Mass (m/z)
150000
200000
ESI Spectrum of Trypsinogen (MW 23983)
M + 15 H+
1599.8
M + 16 H+
M + 14 H+
1499.9
1714.1
M + 13 H+
1845.9
1411.9
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
Reflector
Timed ion detector
selector
Reflector
Laser
Camera
Pumping
Pumping
Linear
detector
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
Mass Sorting (filtering)
Ion
Source
Detection
Ion
Detector
Mass Analyzer
Form ions
(charged molecules)
Sort Ions by Mass (m/z)
Detect ions
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
6130
Poorer
resolution
6140
6150
Mass
6160
6170
ISO:CH3
How is mass resolution calculated?
100
M15.0229
100
90
80
R = M/DM
60
50
FWHM = DM
%
I n te n s i ty
70
40
30
20
10
0
15.01500
15.01820
15.02140
15.02460
M ass (m/z )
15.02780
15.03100
Mass measurement accuracy depends on resolution
High resolution means better mass accuracy
Resolution =18100
8000
15 ppm error
Counts
6000
Resolution = 14200
24 ppm error
4000
Resolution = 4500
2000
55 ppm error
0
2840
2845
2850
Mass (m/z)
2855
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
Single
ion
MS-1
Fragments
MS-2
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
xn-i
vn-i
zn-i
wn-i
-HN--CH--CO--NH--CH--CO--NHRi
CH-R’
ai
R”
bi
ci
di+1
high energy
E=Glu
G=Gly
S=Ser
F=Phe
N=Asn
P=Pro
V=Val
A=Ala
R=Arg
Peptide Fragmentation
=>
E
G
S
F
F
G
E
E
N
P
N
V
A
R
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
Biologist
Who robbed the bank?
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: 5’7”
Weight: 160 lbs
Gender: male
Age: 35-40
Fingerprints
search
DATABASE OF
KNOWN FELONS
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
PEPTIDE MASS
DATABASE
OF KNOWN
PROTEINS
DATABASE SEARCH RESULTS
Police Officer
Mass Spectrometrist
Identifies the robber
Identifies the protein
Anthony J. Felon
bovine carbonic anhydrase
1025. 50
Voyager Spec #1 MC=>AdvBC(32,0.5,0.1)=>NR(1.50)[BP = 1025.5, 26876]
100
Peptide mass fingerprint of Spot A
2.7E+4
Gel coordinates: 16kDa, 4.2 (mwt, pI)
90
80
60
50
0
886.0
1165.6
1445.2
1724.8
Mass (m/z)
2004.4
2211. 10
1994. 99
1786. 82
1708. 75
1277. 71
1308. 66
1107. 56
10
1179. 60
20
1234. 65
30
1544. 69
1341. 63
40
995. 58
% Intensity
70
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.
152
239
326
b4 - 18(+ 1)
y3(+ 1)
y3 - 17(+ 1)
F QL(+ 1), QLF (+ 1)
365. 1
347. 1
y2(+ 1)
F D(+ 1)
229. 2
QL(+ 1) - 28
QL(+ 1)
F Q(+ 1)
b3 - 18(+ 1), A F Q(+ 1) - 17
b2(+ 1)
a2(+ 1)
165. 1
y1 - 17(+ 1)
65
b4(+ 1), QLF D(+ 1) - 28
5.1E+4
y1(+ 1)
F
b1 - 18(+ 1)
Q
L
84. 0
100
90
80
70
60
50
40
30
20
10
0
70. 0
% Intensity
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
0
500
413
Mass (m/z)
720
830
Mass (m/z)
940
973. 7
961. 0
941. 5
y7(+ 1)
b7(+ 1)
819. 9
730. 1
y8(+ 1)
6735.5
714. 8
647. 4
a5(+ 1)
610
A F QLF D(+ 1) - 17, A F QLF D(+ 1) - 18
b5(+ 1)
y4(+ 1)
100
90
80
70
60
50
40
30
20
10
0
500
y4 - 17(+ 1)
% Intensity
Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]
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
381.1
V
I/L
T
1108.13
Partial Sequence
- A-V-I/L-T-
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)
y7
800
700
600
500
y3
b 5 - 2 ( H 2 O)
[M+2H]2+
400
300
I/ L
y6
b 4 - H2 O
739.34
200
S
b 5 - H2 O
y1
100
y9
2 9 4 .2
b 2 - H2 O
y 2 b 3 - H2 O
a2
b1
b2
b3
200
b4
400
Vy8 S
I/L
y10
y 4 b 6 - H2 O
y5
b5
1120.60
y11
600
800
m/z, amu
1000
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.