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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 FranciscoApril 11, 2003

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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 naturalsources, oligonucleotides, drug candidates, peptides,

synthetic organic chemicals, polymers

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Pharmaceutical analysis

Bioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysis

Screening of drug candidatesIdentifying drug targetsBiomolecule characterization

Proteins and peptidesOligonucleotides

Environmental analysisPesticides on foodsSoil and groundwater contamination

Forensic analysis/clinical

Applications of Mass Spectrometry

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How does a mass spectrometer work?

Ion source:makes ions

Massanalyzer:separates

ions

Mass spectrum:presents

information

Sample

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InletIon

source MassAnalyzer Detector DataSystem

High Vacuum System

Mass Spectrometer Block Diagram

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InletIon

source MassAnalyzer Detector DataSystem

High Vacuum System

Mass Spectrometer Block Diagram

Turbo

molecular

pumps

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InletIon

Source MassAnalyzer Detector DataSystem

High Vacuum System

HPLCFlow injection

Sample plate

Sample Introduction

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InletIon

Source MassAnalyzer Detector DataSystem

High Vacuum System

MALDIESI

FAB

LSIMS

EI

CI

Ion Source

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High voltage appliedto 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.)

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hn Laser

1. Sample is mixed with matrix (X)and dried on plate.

2. Laser flash ionizes matrixmolecules.

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

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InletIon

source MassAnalyzer Detector DataSystem

High Vacuum System

Time of flight (TOF)Quadrupole

Ion Trap

Magnetic Sector

FTMS

Mass Analyzer

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Operate under high vacuum (keeps ions from bumpinginto gas molecules)

Actually measure mass-to-charge ratio of ions (m/z)

Key specifications are resolution, mass measurementaccuracy, 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)

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Quadrupole Mass Analyzer

Uses a combination of RF

and DC voltages to operateas a mass filter.

Has four parallel metalrods.

Lets one mass passthrough at a time.

Can scan through all

masses or sit at onefixed mass.

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mass scanning mode

m1m3m4 m2m3

m1

m4

m2

single mass transmission mode

m2 m2 m2 m2

m3

m1

m4

m2

Quadrupoles have variable ion transmission modes

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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.

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Ion Trap Mass Analyzer

Top View

Cut away side view

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InletIon

source MassAnalyzer Detector DataSystem

High Vacuum System

Microchannel PlateElectron Multiplier

Hybrid with photomultiplier

Detector

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+e-

primary ion

e-

e- e-

L

D

-1000V

-100V

L >> D

Ions are detected with a microchannel plate

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InletIon

source MassAnalyzer Detector DataSystem

High Vacuum System

PC

Sun SPARK Station

DEC Station

Data System

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The mass spectrum shows the results

RelativeA

bundance

Mass (m/z)

0

10000

20000

30000

40000

50000 100000 150000 200000

MH+

(M+2H)2+

(M+3H)3+

MALDI TOF spectrum of IgG

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ESI Spectrum of Trypsinogen (MW 23983)

1599.8

1499.9

1714.1

1845.91411.9

1999.62181.6

M + 15 H+

M + 13 H+

M + 14 H+M + 16 H+

m/z Mass-to-charge ratio

H d t t t th i ?

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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

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Voyager-DE STR MALDI TOF

Camera

Laser

Sampleplate

Pumping Pumping

Timed ionselector Reflector

Lineardetector

Extractiongrids

Reflectordetector

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QSTARTM ESI QQ TOF or MALDI QQ TOF

Q1

Ion Mirror

(reflector)

Effective Flight

Path = 2.5 m

Q2

Q0

Sample

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QTRAP: Linear Ion Trap on a Triple Quadrupole

A new type of instrument.

linear ion trap

Exit

Q0 Q1 Q2 Q3

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Inlet

Ionization

Mass Analyzer

Mass Sorting (filtering)

IonDetector

Detection

IonSource

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

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Assigning numerical value to the intrinsic propertyof mass is based on using carbon-12, 12C, as areference point.

One unit of mass is defined as a Dalton (Da).

One Dalton is defined as 1/12 the mass of a singlecarbon-12 atom.

Thus, one 12C atom has a mass of 12.0000 Da.

How is mass defined?

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Isotopes

+Most elements have more than one stable isotope.

For example, most carbon atoms have a mass of 12 Da, but innature, 1.1% of C atoms have an extra neutron, making their mass13 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 theseisotopes, better mass accuracy is achieved.

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Element Mass Abundance

H 1.0078

2.0141

99.985%

0.015

C 12.0000

13.0034

98.89

1.11N 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 ofpeptides

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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

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m/z

4360.45

4361.45

Isotope pattern for a larger peptide (207 C-atoms)

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Mass spectrum of insulin

12C: 5730.61

13C

2 x13

C

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.

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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.

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Average mass

Average mass corresponds

to the centroid of theunresolved 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.

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6130 6140 6150 6160 6170

Poorerresolution

Betterresolution

What if the resolution is not so good?

At lower resolution, the mass measured is the average mass.

Mass

ISO:CH3H i l ti l l t d?

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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

15.0229M

FWHM = DM

R = M/DM

How is mass resolution calculated?

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Mass measurement accuracy depends on resolution

0

2000

4000

6000

8000

Counts

2840 2845 2850 2855

Mass (m/z)

Resolution = 14200

Resolution = 4500

Resolution =1810015 ppm error

24 ppm error

55 ppm error

High resolution means better mass accuracy

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How do we achieve superior massresolution?

Delayed Extraction on a MALDI source

Reflector TOF Mass Analyzer

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Important performance factors

Mass accuracy: How accurate is the massmeasurement?

Resolution: How well separated are the peaksfrom each other?

Sensitivity: How small an amount can beanalyzed?

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What is MSMS?

MS/MS means using two mass analyzers (combinedin one instrument) to select an analyte (ion) from amixture, then generate fragments from it to givestructural information.

Ionsource MS-2MS-1

Mixture ofions

Singleion

Fragments

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What is MS/MS?

MS/MS+

+

++

+

1 peptideselected for

MS/MS

The masses of allthe pieces give anMS/MS spectrum

Peptide

mixture

Have only massesto start

Interpretation of an MSMS spectrum to derive

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Interpretation of an MSMS spectrum to derivestructural information is analogous to solving

a puzzle

+

+

++

+

Use the fragment ion masses as specific pieces ofthe puzzle to help piece the intact molecule backtogether

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-HN--CH--CO--NH--CH--CO--NH-Ri CH-R

ci

zn-i

R

di+1

vn-i wn-ilow energy

high energy

Cleavages Observed in MS/MS of Peptides

ai

xn-i

bi

yn-i

E Glu

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E G S F F G E E N P N V A R

Peptide Fragmentation

175.10246.14345.21459.25556.30670.35799.39

928.43985.451132.521279.591366.621423.641552.69

=>

=

=

=

E=GluG=GlyS=SerF=PheN=Asn

P=ProV=ValA=AlaR=Arg

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Protein Identification

1. Peptide Mass Finger Printing (PMF)from MS data

2. Database search using fragment ion massesfrom MS/MS data

3. Sequence Tagsfrom MS/MS data

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PROBLEM

Bank President

Who robbed the bank?

Biologist

What protein was

isolated?

GATHER EVIDENCE

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Mass Spectrometrist

1. Interview biologist whoisolated 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

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DATABASE SEARCH

Police OfficerHeight: 57

Weight: 160 lbs

Gender: male

Age: 35-40Fingerprints

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

KNOWNFELONS

PEPTIDE MASSDATABASE

OF KNOWNPROTEINS

search search

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DATABASE SEARCH RESULTS

Police Officer

Identifies the robber

Anthony J. Felon

Mass Spectrometrist

Identifies the protein

bovine carbonic anhydrase

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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

%I

ntensity

Voyager Spec #1 MC=>AdvBC (32,0.5,0.1)=>NR (1.50)[B P = 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)

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Mass accuracytolerance = 15 ppm

This means that themass is within

0.015 Da atm/z 1000

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MS/MS t f t ti tid MH+ 1025 5 EAFQLFDR f

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500 610 720 830 940 1050

Mass m/z

0

6735.5

0102030405060708090

100

%I

ntensity

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)

AFQLFD(+1)-

17,A

FQLFD(+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

1020

30

40

50

60

70

80

90

100

%I

ntensity

Stitched PSD=>BC=>SM25=>AdvBC(32,0.5,0.1)[BP = 120.1, 50520]

F

y1(+1)

b2(+1)

FQ(+1)

Qa2(+1)

b4(+1),QLFD(+1)-

28

L b1-

18(+1)

165.1

36

5.1

34

7.1

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

)

b4-1

8(+1)

MS/MS spectrum for tryptic peptide MH+ = 1025.5, EAFQLFDR, fromSpot A. An MS-Tag search using the fragment ions from this spectrumconfirmed the identity of Spot A as myosin light chain.

Sequence Tags from Peptide

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Sequence Tags from PeptideFragmentation 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

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Sequence TAG Example from MS/MS SpectrumPeptide MW = 1345.70

y11

y10

y9

y8

y7

y

6

y5

y4

y3

y2

y1

b1 b2 b3b4 b5

I/ L

a2

294.2b2-H2O

b3-H2O

b4-H2O

b5-H2O

b6-H2O

b5-2(H2O)

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

Sequence Tag search identifies

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Sequence Tag search identifies1 hit (carbonic anhydrase)

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Acknowledgements

We thank the Applied Biosystems Mass Spectrometry ApplicationsLaboratory for allowing the use of some of their slides for thispresentation.