mass spectrometry 101 an introductory lecture on mass spectrometry fundamentals presented to the...

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

Pharmaceutical analysisBioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysisScreening of drug candidatesIdentifying drug targets

Biomolecule characterizationProteins and peptidesOligonucleotides

Environmental analysisPesticides on foodsSoil and groundwater contamination

Forensic analysis/clinical

Applications of Mass Spectrometry

How does a mass spectrometer work?

Ion source:makes ions

Mass analyzer: separates ions

Mass spectrum:presents

information

Sample

InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

Mass Spectrometer Block Diagram

InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

Mass Spectrometer Block Diagram

Turbo molecular pumps

Inlet IonSource

Mass Analyzer Detector

DataSystem

High Vacuum System

HPLCFlow injectionSample plate

Sample Introduction

Inlet IonSource

Mass Analyzer Detector

DataSystem

High Vacuum System

MALDIESIFABLSIMSEICI

Ion Source

High voltage applied to 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.)

h 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

InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

Time of flight (TOF)QuadrupoleIon TrapMagnetic SectorFTMS

Mass Analyzer

¤ 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)

Quadrupole Mass AnalyzerUses 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.

mass scanning mode

m1m3m4 m2

m3

m1

m4

m2

single mass transmission mode

m2 m2 m2 m2m3

m1

m4

m2

Quadrupoles have variable ion transmission modes

Time-of-flight (TOF) Mass Analyzer

+

+

+

+

Source Drift region (flight tube)

dete

ctor

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.

Ion Trap Mass Analyzer

Top View

Cut away side view

InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

Microchannel PlateElectron MultiplierHybrid with photomultiplier

Detector

+e-

primary ion

e-

e- e-L

D

-1000V

-100V

L >> D

Ions are detected with a microchannel plate

InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

PCSun SPARK StationDEC Station

Data System

The mass spectrum shows the results

Re

lativ

e A

bun

dan

ce

Mass (m/z)

0

10000

20000

30000

40000

50000 100000 150000 200000

MH+

(M+2H)2+

(M+3H)3+

MALDI TOF spectrum of IgG

ESI Spectrum of Trypsinogen (MW 23983)

1599.8

1499.9

1714.1

1845.91411.9

1999.6

2181.6

M + 15 H+

M + 13 H+

M + 14 H+M + 16 H+

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: “MALDI-TOF,” “ESI-Quad.”

-Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap

Voyager-DE STR MALDI TOF

Camera

Laser

Sample plate

Pumping Pumping

Timed ion selector Reflector

Linear detector

Extractiongrids

Reflectordetector

QSTARQSTARTMTM ESI QQ TOF or MALDI QQ TOF ESI QQ TOF or MALDI QQ TOF

Q1

Ion Mirror(reflector)

Effective FlightPath = 2.5 m

Q2

Q0

Sample

QTRAP: Linear Ion Trap on a Triple QuadrupoleQTRAP: Linear Ion Trap on a Triple Quadrupole

A new type of instrument….

linear ion trap

Exit

Q0 Q1 Q2 Q3

Inlet

Ionization

Mass Analyzer

Mass Sorting (filtering)

Ion Detector

Detection

Ion Source

• 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

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?

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.

Element Mass AbundanceH 1.0078

2.014199.985%0.015

C 12.000013.0034

98.891.11

N 14.003115.0001

99.640.36

O 15.994916.999117.9992

99.760.040.20

Stable isotopes of most abundant elements of peptides

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”

m/z

4360.45

4361.45

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

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.

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.

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.

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

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

% Int

ensit

yISO:CH3

15.0229M

FWHM = M

R = M/M

How is mass resolution calculated?

Mass measurement accuracy depends on resolution

0

2000

4000

6000

8000

Co

un

ts

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

How do we achieve superior mass resolution?

Delayed Extraction on a MALDI source

Reflector TOF Mass Analyzer

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.

Ion source

MS-2MS-1

Mixture of ions

Single ion

Fragments

What is MS/MS?

MS/MS+

+

+ +

+

1 peptide selected for

MS/MS

The masses of all the pieces give an MS/MS spectrum

Peptidemixture

Have only masses to start

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

-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

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

Peptide Fragmentation

175.10246.14345.21459.25556.30670.35799.39928.43985.45

1132.521279.591366.621423.641552.69

=>

=

=

=

E=GluG=GlyS=SerF=PheN=AsnP=ProV=ValA=AlaR=Arg

Protein Identification

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

2. Database search using fragment ion masses from MS/MS data

3. Sequence Tagsfrom MS/MS data

PROBLEM

Bank President

Who robbed the bank?

Biologist

What protein was isolated?

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

DATABASE SEARCH

Police OfficerHeight: 5’7”

Weight: 160 lbs

Gender: male

Age: 35-40

Fingerprints

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

KNOWN FELONS

PEPTIDE MASS DATABASEOF KNOWN PROTEINS

search search

DATABASE SEARCH RESULTS

Police Officer

Identifies the robber

Anthony J. Felon

Mass Spectrometrist

Identifies the protein

bovine carbonic anhydrase

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

% Int

ensit

yVoyager Spec #1 MC=>AdvBC(32,0.5,0.1)=>NR(1.50)[BP = 1025.5, 26876]

1025.5

0

1341.6

3

1786.8

2

1277.7

1

1179.6

0

1544.6

9

995.58

1234.6

5

1308.6

6

2211.1

0

1708.7

5

1107.5

6

1994.9

9

Peptide mass fingerprint of Spot A

Gel coordinates: 16kDa, 4.2 (mwt, pI)

Mass accuracy tolerance = 15 ppm

This means that the mass is within 0.015 Da at

m/z 1000

500 610 720 830 940 1050

Mass (m/z)

0

6735.5

0102030405060708090

100

% In

tens

ity

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)

AFQL

FD(+

1) -

17,A

FQLF

D(+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

% In

tens

ity

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

y1(+

1)

b2(+

1)

FQ(+

1)

Q

a2(+

1)

b4(+

1),Q

LFD(

+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),A

FQ(+

1) -

17

229.2

FQL(

+1),Q

LF(+

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.

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

Sequence TAG Example from MS/MS Spectrum Peptide MW = 1345.70

y 11

y 10

y 9

y 8

y 7

y 6

y 5y 4

y 3

y 2

y 1

b1 b2 b3 b4 b5

I/ L

a2

294 .2b2 - H2 O

b3 - H2 O

b4 - H2 O

b5 - H2 O

b6 - H2 O

b5 - 2(H2 O)

200 400 600 800 1000 1200m/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 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.