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. 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

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

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

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

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

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

9. Ion Sources make ions from sample molecules (Ions are easier to detect than neutral molecules.)
Electrospray ionization:
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

10. MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate
Laser hn
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+
Grid (0 V)
+/- 20 kV

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

12. 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-of-flight and ion traps are most used.

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

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

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

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

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

21. 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

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
Sample
Linear
Extraction
plate
Reflector
detector
grids
Timed ion
detector
selector
Reflector
Laser
Camera
Pumping
Pumping

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

25. QTRAP: Linear Ion Trap on a Triple Quadrupole
A new type of instrument…. Q0 Q1 Q2 Q3
This instrument has the basic footprint of a triple quadrupole instrument - it’s the same as our small benchtop API The modifications made were the addition of a gridded exit lens and the auxiliary AC- applied dipolar. Also the Q3 is now gold-coated stainless steel rods with two collar lenses, and this is to improve the resolution and peak shape when in LIT mode. Gold-coated rods have less pits on the surface than the stainless steel rods- less imperfect fields means better resolution / sensitivity performance.
Exit
linear ion trap

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

27. 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 Da.

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. Stable isotopes of most abundant elements of peptides

30. Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)
“Monoisotopic mass”
No 13C atoms (all 12C)
One 13C atom
Two 13C atoms

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

32. Mass spectrum of insulin
2 x 13C
13C
12C :
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
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. 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

36. How is mass resolution calculated?
R = M/DM
FWHM = DM

37. High resolution means better mass accuracy
Mass measurement accuracy depends on resolution
High resolution means better mass accuracy
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

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

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.
Mixture of ions
Single ion
Fragments
Ion source
MS-1
MS-2

41. The masses of all the pieces give an MS/MS spectrum
What is MS/MS?
Peptide
mixture
1 peptide
selected for MS/MS +
MS/MS + + + +
The masses of all the pieces give an MS/MS spectrum
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. Cleavages Observed in MS/MS of Peptidesbi
yn-i
zn-i
low energy ai
xn-i
vn-i
wn-i
-HN--CH--CO--NH--CH--CO--NH- Ri
CH-R’ R”
high energy ci
di+1

44. Peptide Fragmentation
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 = = =

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

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

47. GATHER EVIDENCE Mass Spectrometrist
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
3. Analyze peptide mixture by MS to obtain peptide molecular masses!
enzyme

48. Approx. molecular weight: 30,000
DATABASE SEARCH
Mass Spectrometrist
Approx. molecular weight: 30,000
Origin: bovine liver
Peptide mass list from MS analysis: , , , ,
Police Officer
Height: 5’7”
Weight: 160 lbs
Gender: male
Age:
Fingerprints
search
search
DATABASE OF
KNOWN FELONS
PEPTIDE MASS
DATABASE
OF KNOWN
PROTEINS

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

50. 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 Da at
m/z 1000

54. MS/MS spectrum for tryptic peptide MH+ = , 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
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) A V
I/L T
Peptide measured molecular wt =
Partial Sequence
- A-V-I/L-T-
381.1
region region region 3

56. Sequence TAG Example from MS/MS Spectrum Peptide MW = 1345.70y 1 9 8 7 6 5 4 3 2 b I / L a . - H O ( )
200
400
600
800
1000
1200
m/z, amu
100
300
500
700
Sequence Tag (739.34)SVS(I/L)( )
739.34
[M+2H]2+ S V
I/L

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.