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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

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.