1. A New Strategy of Protein Identification in Proteomics Xinmin Yin CS Dept. Ball State Univ.

2. Contents 1.Background. 2.Problems. 3.Proposal. 4.Conclusion.

3. What’s Proteomics ? Use of quantitative protein-level measurement of gene expression to characterize biological process and to decipher the mechanisms of gene expression control.

4. What’s Proteomics ? DNA RNA PROTEIN transcription translation

5. Current Research in Proteomics Quantitative measurement of proteins expressed in a cell * protein identification * protein quantification

6. Experimental Flow Chart of Protein Identification cell proteins peptides mass & sequence data processing

7. Laser Detector1 E1 MS1 E2 Peptides MS2 Detector2 Schematic of MALDI MS/MS Matrix Assisted Laser-induced Desorption Ionization

8. An Example of MALDI MS/MS Spectrum

9. Problem of Scaling up MALDI MS/MS to Proteomics Number of peptides is huge Human genome (~60,000 proteins) 2.6 X 10 6 peptides It will take THREE MONTHS to identify all proteins in a human cell.

10. My Proposal Avoid using MS2 if a peptide can be identified in MS1. Reduce size of peptide database during data analysis To develop a new data analysis method

11. Facts of MALDI MS/MS Experiment 40% peptides are unique in mass when mass accuracy of MALDI is better than 10ppm. Analyzing MS1 spectrum is much easier than analyzing MS2 spectrum. It takes about 1 to 2 min for analysis of a single MS2 spectrum through database.

12. My Proposal Using local protein database for data analysis. Developing a peptide filter. Automating data transfer from MS1 to MS2

13. Local Protein Database Download protein database from public sites. Digest proteins in computer. Select peptides according experimental methods, and store them in memory. Calculate peptide chemical modification. PURPOSE: 1. Make peptide database small according to experimental methods 2. Do online data processing

14. Generating Peptide Database Protein Database Enzyme Digestion Peptide Database Each protein can produce 30 ~ 100 peptides

15. Peptide Filter Identify peptide by mass alone. Sort identified peptides according to proteins. Delete rest peptides of the identified protein from peptide database. Pass unidentified mass information to MS2. PURPOSE: 1. Do online data processing 2. Reduce peptides database size 2. Reduce number of peptides passed to MS2

16. Identifying Peptide by Mass Alone M th M exp MM  M = M th – M exp If (  M <  ) N pep ++ N pep + + N pep : number of mass matched peptides  : mass accuracy of MS1

17. Reducing Size of Peptide Database If (N pep == 1) Peptide is unique; Deleting peptides from the same protein Else Send peptide to MS2 for sequence information.

18. FLOWCHARTFLOWCHART

19. Program Design Computer Language: C++ Classes:Protein Linked list Peptide

20. Conclusion 1. My program will generate a list of peptides from the protein database. 2. Peptide identification is carried out while doing experiment. 3. Data analysis time will be significantly reduced. 4. This program will make identification of proteins from a cell possible and reliable.