Protein Identification

and Peptide Sequencing

by Liquid Chromatography –

Mass Spectrometry

Detlef Schumann, PhDDirector, Proteomics LaboratoryDepartment of Genome Science

May 27, 2005

The Proteomics Problem

Cell state 1

Cell state 2

Why are state 1 and 2

different?

Protein 1

- Protein name: ...- MW: ...- Amino acid sequence: ...- Modifications: ...

Protein 2

-Protein name: ...- MW: ...- Amino acid sequence: ...- Modifications: ...

• • •

Proteomics

The Typical Proteomics Problem

Sample #487 Sample #488

4 7 4 7pI pI

10

200

MW

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

• 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

Basics of Protein Mass Spectrometry

Ion source Mass analyzer Detector

Ion generation Ion separation Ion detection

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

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

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

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

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

1091.620

1799.879

1347.669

1615.722

2143.156

890.612

1978.039 2460.281

1214.658

1504.667

2801.340

0.0

0.5

1.0

1.5

4x10

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000

m/z

Peptide Mass Fingerprinting

Sample: in-gel digested human EF-2

Peptide Mass Fingerprinting Result

Peptide Mass Fingerprinting Result

Tandem Mass Spectrometry (MS/MS) Analysis

eluting peptide mass analysis precursor ion fragmentation fragment mass analysis

Tandem Mass Spectrometry (MS/MS) Analysis

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

b 1 T+

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

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

b-ions y-ions

eluting peptide mass analysis precursor ion fragmentation fragment mass analysis

10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

NL:

1.21E8

Base Peak

400 800 1200 1600m/z

0

100

Rel

ativ

e A

bu

nda

nce

Y121299

Y6689

Y4475 Y122+

650

B101102

B6/Y132+

706

Y7803

B121317

Y8902

B3380

B4493 B9

990

B111204

Y131412

Y5588

Y111202

Y101087

NL5.29E6Base peak

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

400 800 1200 1600

m/z

0

100

Rel

ativ

e A

bund

ance

Y121299

Y6689

Y4475 Y122+

650

B101102

B6/Y132+

706

Y7803

B121317

Y8902

B3380

B4493 B9

990

B111204

Y131412

Y5588

Y111202

Y101087

NL5.29E6Base peak

I/L T I/L V QG D P I/L

Peptide sequence: SYELPDGQVITIGNER

LC-MS/MS Analysis of Protein Digests

LC-MS/MS Analysis Result

LC-MS/MS Analysis Result

Frequently Asked Questions

Short Answer: At least 1 pmol Long Answer: It depends ...

1. How much protein do you need?

- protein staining- protein sequence- protein size- potential post-translational modifications- presence of the protein sequence in the database

Factors:

2. When can I get the results?

Short Answer: In 1-2 weeks Long Answer: It depends ...

- type of requested analysis- amount of protein sample- protein sequence- protein size- potential post-translational modifications- presence of the protein sequence in the database

Factors:

Frequently Asked Questions

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

1 2 3 4 5 6 7 8

Loading (100 ng protein/lane):1 + 2 Ovalbumin (Chicken)3 + 4 Myoglobin (Horse)5 + 6 Cytochrome C (Horse)7 + 8 Serum albumin (Bovine)

Ovalbumin ~ 45 kDa 100 ng ~ 2.2 pmolMyoglobin ~ 17 kDa 100 ng ~ 5.9 pmolCytochrome C ~ 13 kDa 100 ng ~ 7.9 pmolSerum albumin ~ 66 kDa 100 ng ~ 1.5 pmol

The Limitations

1. Protein SizeSmall 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 SequenceProteins 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 ModificationsGlycosylated 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 DatabasesThe 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 Big No-No’s

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

Laboratory AddressProteomics LaboratoryDepartment of Genome Science (ML 0505)Genome Research InstituteUniversity of CincinnatiBuilding B, Room 1312180 East Galbraith RoadCincinnati, Ohio 45237Tel: 513/558-8950Fax: 513/558-5061Email: detlef.schumann@uc.edu

Staff Members• Detlef Schumann• Wendy Dominick • Michael Wyder• Margaret Minges

Contact Information