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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.

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slide1

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

slide2

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

slide3

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
slide4

Ion source:makes ions

How does a mass spectrometer work?

Sample

Mass analyzer: separates ions

Mass spectrum:presents information

slide5

Mass Spectrometer Block Diagram

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

slide6

Mass Spectrometer Block Diagram

Turbo molecular pumps

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

slide7

Sample Introduction

High Vacuum System

Ion

Source

Mass

Analyzer

Data

System

Inlet

Detector

HPLC

Flow injection

Sample plate

slide8

Ion Source

High Vacuum System

Ion

Source

Mass

Analyzer

Data

System

Inlet

Detector

MALDI

ESI

FAB

LSIMS

EI

CI

slide9

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

Partialvacuum

Sample Inlet Nozzle

(Lower Voltage)

Pressure = 1 atmInner tube diam. = 100 um

MH+

N2

+

+

+

+

+

+

+

+

+

+

+

+

MH2+

+

+

+

+

+

+

+

+

+

+

+

+

Sample in solution

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

N2 gas

+

+

+

+

+

MH3+

High voltage applied

to metal sheath (~4 kV)

Charged droplets

Electrospray ionization:

slide10

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

slide11

Mass Analyzer

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Time of flight (TOF)

Quadrupole

Ion Trap

Magnetic Sector

FTMS

slide12

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.
slide13

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.
slide14

m1

m2

m4

m3

m2

m1

m4

m3

mass scanning mode

m1

m2

m2

m2

m2

m2

m4

m3

single mass transmission mode

Quadrupoles have variable ion transmission modes

slide15

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.
slide16

Ion Trap Mass Analyzer

Top View

Cut away side view

slide17

Detector

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

Microchannel Plate

Electron Multiplier

Hybrid with photomultiplier

ions are detected with a microchannel plate
Ions are detected with a microchannel plate

primary ion

-

1000V

+

-

e

-

e

L

-

e

-

e

-

100V

D

L >> D

slide19

Data System

High Vacuum System

Ion

source

Mass

Analyzer

Data

System

Inlet

Detector

PC

Sun SPARK Station

DEC Station

slide20

40000

30000

20000

10000

0

The mass spectrum shows the results

MALDI TOF spectrum of IgG

MH+

Relative Abundance

(M+2H)2+

(M+3H)3+

50000

100000

150000

200000

Mass (m/z)

slide21

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

slide22

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
Voyager-DE STR MALDI TOF

Sample

Linear

Extraction

plate

Reflector

detector

grids

Timed ion

detector

selector

Reflector

Laser

Camera

Pumping

Pumping

slide24

QSTARTM ESI QQ TOF or MALDI QQ TOF

Sample

Q0

Q1

Q2

Effective Flight

Path = 2.5 m

Ion Mirror

(reflector)

slide25

QTRAP: Linear Ion Trap on a Triple Quadrupole

A new type of instrument….

Q0

Q1

Q2

Q3

Exit

linear ion trap

slide26

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

0

1330

1340

1350

Mass Spectrum

slide27

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.

slide28

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.

slide30

1981.84

1982.84

1983.84

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

slide31

4361.45

4360.45

m/z

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

mass spectrum of insulin
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

When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

average mass
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.

slide35

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

slide37

Resolution =18100

8000

6000

Resolution = 14200

Counts

4000

Resolution = 4500

2000

0

2840

2845

2850

2855

Mass (m/z)

Mass measurement accuracy depends on resolution

High resolution means better mass accuracy

15 ppm error

24 ppm error

55 ppm error

slide38

How do we achieve superior mass resolution?

Reflector TOF Mass Analyzer

Delayed Extraction on a MALDI source

slide39

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?
slide40

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

slide41

What is MS/MS?

1 peptide

selected for MS/MS

Peptide

mixture

+

+

MS/MS

+

+

+

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

Have only masses to start

slide42

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
Cleavages Observed in MS/MS of Peptides

yn-i

xn-i

ai

bi

low energy

high energy

zn-i

vn-i

wn-i

-HN--CH--CO--NH--CH--CO--NH-

Ri

CH-R’

R”

ci

di+1

slide44

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

1132.52

1279.59

1366.62

1423.64

1552.69

=

=

=

slide45

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?

PROBLEM
gather evidence
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!

Police Officer

1. Interview witnesses

2. Dust for fingerprints

GATHER EVIDENCE

enzyme

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 SEARCH

search

search

DATABASE OF

KNOWN FELONS

PEPTIDE MASS

DATABASE

OF KNOWN

PROTEINS

database search results
Police Officer

Identifies the robber

Anthony J. Felon

Mass Spectrometrist

Identifies the protein

bovine carbonic anhydrase

DATABASE SEARCH RESULTS
slide50

Peptide mass fingerprint of Spot A

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

slide51

Mass accuracy tolerance = 15 ppm

This means that the mass is within 0.015 Da at

m/z 1000

slide54

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.

slide55

Peptide measured molecular wt= 1927.2

A

V

I/L

T

1108.13

Partial Sequence

- A-V-I/L-T-

381.1

region 1region 2region 3

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)

1546.11

sequence tag example from ms ms spectrum peptide mw 1345 70

Sequence Tag (739.34)SVS(I/L)(1120.60)

y

7

800

700

600

[M+2H]2+

500

y

3

b

-

2

(

H

O

)

5

2

400

I

/

L

300

y

b

-

H

O

6

4

2

y

9

739.34

200

S

V

S

I/L

y

b

-

H

O

8

5

2

y

1

y

2

9

4

.

2

1

0

b

-

H

O

6

2

100

y

4

b

-

H

O

1120.60

2

2

y

b

-

H

O

5

y

3

2

2

a

2

b

b

b

5

b

b

4

1

2

3

y

1

1

200

400

600

800

1000

1200

m/z, amu

Sequence TAG Example from MS/MS Spectrum Peptide MW = 1345.70
slide58

Acknowledgements

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