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Protein Identification and Peptide Sequencing by Liquid Chromatography – Mass Spectrometry. Detlef Schumann, PhD Director, Proteomics Laboratory Department of Genome Science May 27, 2005. Cell state 1. Protein 1 Protein name: ... MW: ... Amino acid sequence: ...

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Protein Identification and Peptide Sequencing by Liquid Chromatography – Mass Spectrometry

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Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Protein Identification

and Peptide Sequencing

by Liquid Chromatography –

Mass Spectrometry

Detlef Schumann, PhD

Director, Proteomics Laboratory

Department of Genome Science

May 27, 2005


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Cell state 1

  • Protein 1

  • Protein name: ...

  • MW: ...

  • Amino acid sequence: ...

  • Modifications: ...

  • Protein 2

  • Protein name: ...

  • MW: ...

  • Amino acid sequence: ...

  • Modifications: ...

Proteomics

Why are state 1 and 2 different?

Cell state 2

The Proteomics Problem


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

The Typical Proteomics Problem

Sample #488

Sample #487

MW

200

10

4

7

4

7

pI

pI


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

The Proteomics Laboratory at the GRI

Electrophoresis Laboratory

  • 1-D gel electrophoresis (small format)

  • 2-D gel electrophoresis (small and large format)

  • Silver staining and Coomassie staining

  • Imaging densitometry of protein gels

  • Comparative 2-D gel data analysis

  • Western blotting (small format gels)

  • HPLC separation of protein mixtures

Mass Spectrometry Laboratory

  • Peptide mass fingerprinting

  • LC-MS/MS analysis

  • Analysis of protein modifications

  • Purity analysis of recombinant proteins/synthetic peptides

  • Purity analysis of oligonucleotides


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Detector

Ion source

Mass analyzer

Ion generation

Ion separation

Ion detection

F. Lottspeich and H. Zorbas, Bioanalytik 1998, Spektrum Akad. Verlag

Basics of Protein Mass Spectrometry

  • Mass spectrometry determines the molecular weight of chemical compounds by separating molecular ions in a vacuum according to their mass-to-charge ratio (m/z)

  • Ions are generated by induction of either the loss or the gain of a charge (protonation, deprotonation or electron injection)

  • Generated ions can be fragmented in the vacuum, and the resulting sub-fragments can provide information about the structure of a compound


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

1. Bruker Biflex III MALDI-TOF mass spectrometer

• mid fmole protein/peptide analysis

• protein identification using peptide mass fingerprinting

• oligonucleotide mass/purity analysis

• biomarker analysis

2. Finnigan LCQ Deca XP Max ESI mass spectrometer

• coupled to Dionex Ultimate nanoflow 2-D HPLC

• low fmole peptide analysis

• protein identification using LC-MS/MS peptide sequencing

3. PE Sciex API 3000 ESI mass spectrometer

• low pmole/high fmole peptide/metabolite analysis

• identification of post-translational modifications

• peptide and metabolite quantitation studies

Mass Spectrometry Instrumentation at the GRI


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Protein Identification by Mass Spectrometry

1. Peptide Mass Fingerprinting

  • protease digestion of protein spots/bands

  • peptide extraction

  • sample spotting on target plate

  • mass measurement of peptide ions by MALDI-TOF MS or LC-MS

  • data base search using generated mass list

  • protein identification based on ≥ 4 matched peptide masses

2. Peptide Sequencing

  • protease digestion of protein spots/bands

  • peptide extraction

  • RP-LC separation of peptides

  • mass measurement and fragmentation analysis of peptide ions

  • data base search using parent mass and fragment mass data

  • protein identification based on ≥ 2 matched peptides


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Peptide Mass Fingerprinting

Sample:

in-gel digested human EF-2


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Peptide Mass Fingerprinting Result


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Peptide Mass Fingerprinting Result


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

eluting peptide

mass analysis

precursor ion

fragmentation

fragment mass analysis

Tandem Mass Spectrometry (MS/MS) Analysis


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

eluting peptide

mass analysis

precursor ion

fragmentation

fragment mass analysis

T E S T P E P T I D E+

b-ions

y-ions

TESTPEPTIDE+ y11

ESTPEPTIDE+ y10

STPEPTIDE+ y 9

TPEPTIDE+ y 8

PEPTIDE+ y 7

EPTIDE+ y 6

PTIDE+ y 5

TIDE+ y 4

IDE+ y 3

DE+ y 2

E+ y 1

T E S T + P E P T I D E+

b 1T+

b 2 TE+

b 3 TES+

b 4 TEST+

b 5 TESTP+

b 6 TESTPE+

b 7 TESTPEP+

b 8 TESTPEPT+

b 9 TESTPEPTI+

b10 TESTPEPTID+

b11 TESTPEPTIDE+ - H2O

Tandem Mass Spectrometry (MS/MS) Analysis


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

NL:

100

1.21E8

Base Peak

Y12

1299

100

NL

5.29E6

Base peak

80

Y6

689

B10

1102

B6/Y132+

706

Y4

475

60

Y10

1087

B12

1317

Relative Abundance

Y122+

650

B11

1204

B4

493

B3

380

Y7

803

B9

990

Relative Abundance

Y8

902

Y13

1412

Y11

1202

Y5

588

40

0

400

800

1200

1600

m/z

20

0

60

35

10

20

45

15

30

55

25

50

40

Time (min)

LC-MS/MS Analysis of Protein Digests

Base peak chromatogram of the LC-MS/MS analysis of a protein digest from a silver stained 2D gel spot, the

insert showing the MS/MS spectrum for the actin peptide SYELPDGQVITIGNER as identified by SEQUEST


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

I/L

T

I/L

V

QG

D

P

I/L

Y12

1299

NL

5.29E6

Base peak

100

Y6

689

B10

1102

B6/Y132+

706

Relative Abundance

Y4

475

Y10

1087

B12

1317

Y122+

650

B11

1204

B4

493

B3

380

B9

990

Y7

803

Y13

1412

Y8

902

Y11

1202

Y5

588

0

400

800

1200

1600

m/z

LC-MS/MS Analysis of Protein Digests

Peptide sequence: SYELPDGQVITIGNER


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

LC-MS/MS Analysis Result


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

LC-MS/MS Analysis Result


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Factors:

- protein staining

- protein sequence

- protein size

- potential post-translational modifications

- presence of the protein sequence in the database

Factors:

- type of requested analysis

- amount of protein sample

- protein sequence

- protein size

- potential post-translational modifications

- presence of the protein sequence in the database

Frequently Asked Questions

1. How much protein do you need?

Short Answer: At least 1 pmol

Long Answer: It depends ...

2. When can I get the results?

Short Answer: In 1-2 weeks

Long Answer: It depends ...


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

1

2

3

4

5

6

7

8

Loading (100 ng protein/lane):

1 + 2Ovalbumin (Chicken)

3 + 4Myoglobin (Horse)

5 + 6Cytochrome C (Horse)

7 + 8 Serum albumin (Bovine)

Ovalbumin ~ 45 kDa100 ng ~ 2.2 pmol

Myoglobin ~ 17 kDa100 ng ~ 5.9 pmol

Cytochrome C ~ 13 kDa 100 ng ~ 7.9 pmol

Serum albumin ~ 66 kDa100 ng ~ 1.5 pmol

Frequently Asked Questions

3. I saw a dark band/spot on the gel. Why did we get no results?


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

1. Protein Size

Small proteins ( 10 kDa) or large proteins ( 150 kDa) are more challenging to digest and analyze because they generate few peptides (small proteins) or show increased resistance to proteases (large proteins).

2. Protein Sequence

Proteins are typically digested using trypsin (K/R cleavage); the distribution of these AA dictates the size and the detectability of the generated peptides.

3. Post-translational Modifications

Glycosylated proteins show high resistance to proteases; certain post-translational modifications (e.g. phosphorylation) decrease the detectability of the modified peptide using the standard protein mass spectrometry techniques.

4. Protein Sequence Databases

The database search algorithms compare the generated spectra with theoretical digests of proteins in protein sequence databases; the positive identification of the analyzed protein depends on the presence of its sequence in those databases.

The Limitations


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

1. Detergents

Detergents used for extraction and purification of proteins, when not completely removed, can cause signal suppression and decreased detectability of peptides in the mass spectrometry analysis

2. Contaminants

In-gel digests of low abundance samples are very sensitive to the presence of contaminants, particularly contaminating proteins. The handling of samples/gels with gloves is absolutely necessary and the use of designated equipment for specific separation and staining protocols is highly recommended.

3. Formaldehyde or Glutaraldehyde Fixation in Silver Staining

While increasing the staining sensitivity, these fixation steps result in a covalent modification and cross-linking of proteins, which can result in decreased digestion efficiency.

The Big No-No’s


Protein identification and peptide sequencing by liquid chromatography mass spectrometry

Contact Information

  • Laboratory Address

    • Proteomics Laboratory

    • Department of Genome Science (ML 0505)

    • Genome Research Institute

    • University of Cincinnati

    • Building B, Room 131

    • 2180 East Galbraith Road

    • Cincinnati, Ohio 45237

    • Tel: 513/558-8950

    • Fax: 513/558-5061

    • Email: [email protected]

  • Staff Members

    • • Detlef Schumann

    • • Wendy Dominick

    • • Michael Wyder

    • • Margaret Minges


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