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. The Proteomics Problem 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

3. The Typical Proteomics Problem Sample #487 Sample #488 47 47 pI 10 200 MW

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. 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 sourceMass analyzer Detector Ion generationIon separation Ion detection F. Lottspeich and H. Zorbas, Bioanalytik 1998, Spektrum Akad. Verlag

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 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

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

11. Tandem Mass Spectrometry (MS/MS) Analysis eluting peptidemass analysisprecursor ionfragmentationfragment mass analysis

12. 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 + b 10 TESTPEPTID + b 11 TESTPEPTIDE + - H 2 O TESTPEPTIDE + y 11 ESTPEPTIDE + y 10 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 peptidemass analysisprecursor ionfragmentationfragment mass analysis

13. 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. 40080012001600 m/z 0 100 Relative Abundance Y12 1299 Y6 689 Y4 475 Y12 2+ 650 B10 1102 B6/Y13 2+ 706 Y7 803 B12 1317 Y8 902 B3 380 B4 493 B9 990 B11 1204 Y13 1412 Y5 588 Y11 1202 Y10 1087 NL 5.29E6 Base peak I/LT VQGDPI/L Peptide sequence: SYELPDGQVITIGNER LC-MS/MS Analysis of Protein Digests

15. LC-MS/MS Analysis Result

17. Frequently Asked Questions Short Answer: At least 1 pmolLong 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 weeksLong 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:

18. Frequently Asked Questions 3. I saw a dark band/spot on the gel. Why did we get no results? 12345678 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

19. The Limitations 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.

20. The Big No-No’s 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.

21. 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 Contact Information