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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. 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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  1. Protein Identification and Peptide Sequencing by Liquid Chromatography – Mass Spectrometry Detlef Schumann, PhD Director, Proteomics Laboratory Department of Genome Science May 27, 2005

  2. 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

  3. The Typical Proteomics Problem Sample #488 Sample #487 MW 200 10 4 7 4 7 pI pI

  4. 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

  5. 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

  6. 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

  7. 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

  8. Peptide Mass Fingerprinting Sample: in-gel digested human EF-2

  9. Peptide Mass Fingerprinting Result

  10. Peptide Mass Fingerprinting Result

  11. eluting peptide mass analysis precursor ion fragmentation fragment mass analysis Tandem Mass Spectrometry (MS/MS) Analysis

  12. 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

  13. 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

  14. 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

  15. LC-MS/MS Analysis Result

  16. LC-MS/MS Analysis Result

  17. 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 ...

  18. 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 pmol Myoglobin ~ 17 kDa 100 ng ~ 5.9 pmol Cytochrome C ~ 13 kDa 100 ng ~ 7.9 pmol Serum albumin ~ 66 kDa 100 ng ~ 1.5 pmol Frequently Asked Questions 3. I saw a dark band/spot on the gel. Why did we get no results?

  19. 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

  20. 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

  21. 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: detlef.schumann@uc.edu • Staff Members • • Detlef Schumann • • Wendy Dominick • • Michael Wyder • • Margaret Minges

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