1. Proteomics I Mass Spectrometry Functional Genomics by Mass Spectrometry (Andersen and Mann, 2000) FEBS Letters 480, 25-31 optional

2. Proteomics II Yeast Two Hybrid Toward a Protein-Protein Map of the Budding Yeast (Ito et al., 2000) PNAS 97(3), 1143-1147 optional

3. Mass Spectrometry Molecules to be analyzed, referred to as analytes are first ionized (usually in a vacuum), Newly charged molecules are introduced into an electric and/or magnetic field in gas phase, Their path through the field is a function of the mass to charge ratio m/z, m/z of the ionized species can be used to deduce the mass of the analyte with high precision.

4. Biological Samples....bringing polypeptides and nucleic acids to the gas phase usually degrades the molecules, matrix assisted laser desorption/ionization mass spectrometry MALDI-MS 1988 electrospray ionization mass spectrometry ESI-MS 2002 Nobel Prize on Chemistry, Fenn & Tanaka

5. Electrospray Ionization Mass Spectrometry ESI-MS Peptides analytes, in solution, are passed through a charged needle that is kept at high electrical potential, –the peptides are ionized, –this disperses the the solution into a fine spray, –the solvent quickly evaporates, – peptides now in gas phase, Enter mass spectrometer for mass fingerprinting, or Peptide Sequencing.

6. ESI-MS

7. Multi-protein Complexes “mud pit” analysis ?...i.e. nuclear pore complexes,...i.e. cellulose synthase complexes,...i.e. spindle pole apparati,...i.e. proteins involved in the spliceosome, …etc. etc. etc.

9. Post Transcriptional Modification eukaryotes Differential splicing can alter the final proteins function.

10. 5 ’ mRNA Cap m 3 G Cap

11. Eukaryotic Intron Excision (sequence is important)

12. Spliceosomes confer sequence specificity... small nuclear RNAs (snRNAs): –RNA molecules that act as function in spliceosomes, …work in concert with > 100 proteins to facilitate intron identification and removal, –snRNPs: RNA/Protein structures.

13. U1 and U2 snRNAs U1 binds to the 5 ’ exon/intron junction. U2 binds to the adenosine region at the branch site. Psuedouradine (  Think about the required specificity for intron identification in cells.

14. Eukaryotic Intron Excision

15. Base-pairing specificity of the snRNAs give the cell control of intron excision. U2 and U6, snRNAs proteins not shown

16. U1 snRNP(s)

17. Isolate Spliceosome Complex Anti-m 3 G Antibody Chromotography...RNA processing includes the capping of the 5’ end by 7- methylguanosine triphosphate, …antibodies are available for this molecule, …all molecules with a capped RNA can be co- precititated. Histidine Tag Snp1 Nickel Nitrilotriacetic Acid (Ni-NTA) Affinity Chromotography...Snp1 has been identified as a U1 specific snRNP,...the functional Snp1, in the experiment (yeast), is carried on a plasmid,...it was modified by a hexahistidine addition to the N-terminus,...the exposed his-tag binds to nickel on a chromatography column, is washed off with imidazole.

18. Separate Ni-NTA Eluate... SDS-PAGE fractionation yields 20 proteins, bands are in-gel digested, extracted, sent to MS-MS for ID.

19. Glycerol Gradient

20. b-type ions (  -amino)y”-type ions (  -carboxyl)

21. MS-MS A. fragment at 756.9 was selected for sequencing, B. LEEL sequenced, C. AELEELEEFEFK unique match in the database. Other partially sequenced peptides correspond (underlined).

22. Whole Complex De-convolution...peptides can be isolated and from heterogenous samples, sequenced and ID’d. * Prp39p (106 aa peptide) Prp40p (41 aa peptide)

23. Found Players All of the known U1-specific proteins found to date were identified, An ortholog to human U1-C was found in yeast, that had not been been previously identified, Four novel proteins were also identified.

24. Establishment of Principle...so, in one fell swoop, years of molecular genetic and biochemical research was replicated, and...a U1-C protein, not previously detected, was identified as a component in the system, and...four novel proteins have been ID’d, –not necessarily components, –but excellent research leads.

25. Use of Principle...in one subsequent experiment with isolated human spliceosome, –70 spliceosome protein spots were analyzed, –19 novel splicing factors were identified, and...all 19 could be cloned directly via EST libraries, and...in vivo conformation of the role of these factors in splicing was obtained through co-localization studies using green fluorescence protein (GFP).

26. Organelle (nucleolar proteins)

27. Spliceosome Analysis Yeast Two-Hybrid Nature Genetics, 1997, 16 pp 277 - 282 Proteomics II

28. GAL4 Transcription Activator BD: Binding Domain, AD: Activation Domain. GAL4

29. Strategy Bait: 10 spliceosome proteins, Targets: genomic library, Selections: His + and lacZ +, Sequence: identify ORFs, 2 nd Round: use identified preys as bait, Repeat.

30. Results

32. Wednesday Questions. Bioinformatics II