Mass spectroscopy

1. 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

2. 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

3. 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
Applications of Mass Spectrometry

4. How does a mass spectrometer work?
Ion source:
makes ions
Mass
analyzer:
separates
ions
Mass spectrum:
presents
information
Sample

5. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram

6. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram
Turbo
molecular
pumps

7. Inlet Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
HPLC
Flow injection
Sample plate
Sample Introduction

8. Inlet Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
MALDI
ESI
FAB
LSIMS
EI
CI
Ion Source

9. High voltage applied
to metal sheath (~4 kV)
Sample Inlet Nozzle
(Lower Voltage)
Charged droplets
++
+
+
+
+
+
+
++
+
+
+
+
++
+
+ +
++
+
++
+
++
+++
+
+
+
+
+
+
+
+
++
+
++
++
+
MH+
MH3
+
MH2
+
Pressure = 1 atm
Inner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partial
vacuum
Electrospray ionization:
Ion Sources make ions from sample molecules
(Ions are easier to detect than neutral molecules.)

10. hn
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

11. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Time of flight (TOF)
Quadrupole
Ion Trap
Magnetic Sector
FTMS
Mass Analyzer

12. ¤ 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)

13. 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.

14. mass scanning mode
m1m3m4 m2
m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2
m3
m1
m4
m2
Quadrupoles have variable ion transmission modes

15. 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.

16. Ion Trap Mass Analyzer
Top View
Cut away side view

17. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Microchannel Plate
Electron Multiplier
Hybrid with photomultiplier
Detector

18. +
e-
primary ion
e-
e- e-
L
D
-1000V
-100V
L >> D
Ions are detected with a microchannel plate

19. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
PC
Sun SPARK Station
DEC Station
Data System

20. The mass spectrum shows the results
RelativeAbundance
Mass (m/z)
0
10000
20000
30000
40000
50000 100000 150000 200000
MH+
(M+2H)2+
(M+3H)3+
MALDI TOF spectrum of IgG

21. ESI Spectrum of Trypsinogen (MW 23983)
1599.8
1499.9
1714.1
1845.9
1411.9
1999.6
2181.6
M + 15 H+
M + 13 H+
M + 14 H+
M + 16 H+
m/z Mass-to-charge ratio

22. 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

23. Voyager-DE STR MALDI TOF
Camera
Laser
Sample
plate
Pumping Pumping
Timed ion
selector Reflector
Linear
detector
Extraction
grids
Reflector
detector

24. QSTARTM
ESI QQ TOF or MALDI QQ TOF
Q1
Ion Mirror
(reflector)
Effective Flight
Path = 2.5 m
Q2
Q0
Sample

25. QTRAP: Linear Ion Trap on a Triple Quadrupole
A new type of instrument….
linear ion trap
Exit
Q0 Q1 Q2 Q3

26. Inlet
Ionization
Mass Analyzer
Mass Sorting (filtering)
Ion
Detector
Detection
Ion
Source
• Solid
• Liquid
• Vapor
Detect ions
Form ions
(charged molecules)
Sort Ions by Mass (m/z)
1330 1340 1350
100
75
50
25
0
Mass Spectrum
Summary: acquiring a mass spectrum

27. 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?

28. 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.

29. Element Mass Abundance
H 1.0078
2.0141
99.985%
0.015
C 12.0000
13.0034
98.89
1.11
N 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 of
peptides

30. 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”

31. m/z
4360.45
4361.45
Isotope pattern for a larger peptide (207 C-atoms)

32. 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.

33. 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.

34. 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.

35. 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

36. 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
ISO:CH3
15.0229
M
FWHM = DM
R = M/DM
How is mass resolution calculated?

37. Mass measurement accuracy depends on resolution
0
2000
4000
6000
8000
Counts
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

38. How do we achieve superior mass
resolution?
Delayed Extraction on a MALDI source
Reflector TOF Mass Analyzer

39. 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?

40. 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

41. What is MS/MS?
MS/MS
+
+
+ +
+
1 peptide
selected for
MS/MS
The masses of all
the pieces give an
MS/MS spectrum
Peptide
mixture
Have only masses
to start

42. 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

43. -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

44. E G S F F G E E N P N V A R
Peptide Fragmentation
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
=>
=
=
=
E=Glu
G=Gly
S=Ser
F=Phe
N=Asn
P=Pro
V=Val
A=Ala
R=Arg

45. 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

46. PROBLEM
Bank President
Who robbed the bank?
Biologist
What protein was
isolated?

47. 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

48. DATABASE SEARCH
Police Officer
Height: 5’7”
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
DATABASE OF
KNOWN FELONS
PEPTIDE MASS
DATABASE
OF KNOWN
PROTEINS
search search

49. DATABASE SEARCH RESULTS
Police Officer
Identifies the robber
Anthony J. Felon
Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase

50. 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
%Intensity
VoyagerSpec#1MC=>AdvBC(32,0.5,0.1)=>NR(1.50)[BP=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)

51. Mass accuracy
tolerance = 15 ppm
This means that the
mass is within
0.015 Da at
m/z 1000

54. 500 610 720 830 940 1050
Mass(m/z)
0
6735.5
0
10
20
30
40
50
60
70
80
90
100
%Intensity
StitchedPSD=>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,AFQLFD(+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
%Intensity
StitchedPSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP=120.1,50520]F
y1(+1)
b2(+1)
FQ(+1)
Q
a2(+1)
b4(+1),QLFD(+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),AFQ(+1)-17
229.2
FQL(+1),QLF(+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.

55. 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

56. Sequence TAG Example from MS/MS Spectrum
Peptide MW = 1345.70
y 1 1
y 1 0
y 9
y 8
y 7
y 6
y 5
y 4
y 3
y 2
y 1
b1 b2 b3 b4 b5
I/ L
a2
2 9 4 .2
b2 - H2 O
b3 - H2 O
b4 - H2 O
b5 - H2 O
b6 - H2 O
b5 - 2 ( H2 O)
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

57. Sequence Tag search identifies
1 hit (carbonic anhydrase)

58. Acknowledgements
We thank the Applied Biosystems Mass Spectrometry Applications
Laboratory for allowing the use of some of their slides for this
presentation.