1. Click to add Text Sample Preparation for Mass Spectrometry Sermin Tetik, PhD Marmara University July 2015, New Orleans

2. Click to add Text An overwiev Introduction Sample Lysis and Protein Extraction Protein Enrichment Protein Quantitation and Staining Peptide Clean Up

3. Click to add Text Introduction Proper sample preparation for MS-based analysis is critical step in the proteomics workflow.

4. Click to add Text Introduction Mass spectrometry allows for the analysis of samples ranging in mass from 50-300,000 daltons, in attomole through nanomole quantites.

5. Click to add Text Introduction The most successful proteomics Labs Sample Preparation InstrumentationSoftware

6. Click to add Text Proper Sample Preparation Means Better Results Proper Sample Preparation Means Better Results Two significant problems in MS analysis First, the ionization techniques used for large molecules work Second, mass spectrum from a complex mixture is very difficult to fully analyze

7. Click to add Text Proper Sample Preparation Means Better Results Proper Sample Preparation Means Better Results

8. Click to add Text Discovery vs. Targeted Proteomic Analysis Discovery vs. Targeted Proteomic Analysis A succsessful proteomics experiment requires integration of the right sample preparation, instrumentation and software.

9. Click to add Text Discovery vs. Targeted Proteomic Analysis Discovery vs. Targeted Proteomic Analysis Discovery proteomics maximizes protein identification. Targeted proteomics strategies limit the number of features.

10. Click to add Text Targeted Proteomic Analysis Targeted Proteomic Analysis Targeted proteomics experiments are quantified less than 100 proteins with very high precision, sensitivity, specifity and throughput....are used in Pharmaceutical and Diagnostic applications

11. Click to add Text Mass Specrometry Workflows Mass Specrometry Workflows

12. Click to add Text Mass Specrometry Workflow Mass Specrometry Workflow

13. Click to add Text Sample Lysis and Protein Extraction

14. Click to add Text All living organisms contain proteolytic enzymes for protein catabolism. Sample Lysis and Protein Extraction

15. Click to add Text Phosphatases are another class of enzymes that are liberated during cell lysis. 80% occur'ng on serine 20% occur'ng on threonine 1% on threonine residues Sample Lysis and Protein Extraction

16. Click to add Text Sample Lysis and Protein Extraction

17. Click to add Text Abundant Protein Depletion

18. Click to add Text Abundant Protein Depletion

19. Click to add Text Abundant Protein Depletion

20. Click to add Text Protein Enrichment Biochemical activity Post-translaional modification (PTM)

21. Click to add Text Protein Enrichment Shematic summary of a standard immunoprecipitation assay

22. Click to add Text Protein Enrichment Recent advances in MS technology, in combination with phosphoprotein enrichment using immobilized metal affinity chromatography, have resulted in greater solution of the phosphoproteome.

23. Click to add Text Active Site Protein Labeling and Enrichment Western blot and MS workflows for targeted capture and analysis of enzymes

24. Click to add Text Protein Clean Up DialysisDesaltingConcentrating Salts or detergents

25. Click to add Text Protein Clean Up

26. Click to add Text Protein Quantitation and Staining In-gel digestion SDS-PAGEextracting

27. Click to add Text Protein Quantitation and Staining

28. Click to add Text Protein Quantitation and Staining

29. Click to add Text Protein Digestion

30. Click to add Text Protein Digestion

31. Click to add Text Protein Digestion

32. Click to add Text Protein Digestion

33. Click to add Text Protein Digestion

34. Click to add Text Low-abundant Proteins Post Translationally Modified Peptides 1. Enrichment 2. Clean up Peptide Enrichment

35. Click to add Text Peptide Enrichment

36. Click to add Text Peptide Enrichment

37. Click to add Text Peptide Enrichment

38. Click to add Text Peptide clean up C18 spin Column C4 column Tips

39. Click to add Text Peptide clean up

40. Click to add Text Thank You