PRELIMINARY RESULTS OF THE MRM ANALYSIS

1. PRELIMINARY RESULTS OF THE MRM ANALYSIS sHPP CHROMOSOME 16 MEETING August, 28, 2012 La Cristalera, Miraflores de la Sierra

2. INDEX • ASSIGNED PROTEINS AND PROBLEMS • MATERIALS AND METHODS • RESULTS: • ENTIRE EXTRACTS OF THE 3 CELL LINES SAMPLES • CONCLUSIONS • THE NOMO1 CASE

3. KNOWN PROTEINS ASSIGNED TO THE UCM-PCM PROTEOMICS UNIT

4. MATERIALS AND METHODS

5. SEVERAL TYPES OF SAMPLES • 3 CELL LINES: • Ramos cell line (Ph. D. Fuentes) • MCF7 cell line (Ph. D. Canals) • CCD18 cell line (Ph. D. Casals) • For MCF7 cell line, the protein quantity (Bradford method) was approx. the half than it should be. • IN SOLUTION DIGESTIONS OF THE PROTEIN EXTRACT: • The in solution digestion for the 3 lines were performed. • Own lab protocol.

6. PROTEINS AND PEPTIDES SEARCH • PROTEIN SEQUENCES USING SWISSPROT ACCESSION NUMBER . (http://www.uniprot.org/) • ENSEMBL ENTRANCES. (http://www.ensembl.org/index.html) • NCBI BLAST AND SKYLINE TO SEARCH PROTEOTYPIC PEPTIDES. • (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) • https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view • MATCH THE SELECTED PEPTIDES IN LITERATURE: • JURKAT CELL TYPE RESULTS: provided by the CNB Proteomics Unit. • MANN’S ARTICLE: provided by Ph. D. I. Casals (CIB). • SRM ATLAS DATA. • GPM AND NIST SPECTRA DATABASES FOR SKYLINE SOFTWARE. • MRM PILOT.

7. EQUIPMENTS AND SOFTWARES • PREDICTOR SOFTWARES: • SRM ATLAS(http://www.srmatlas.org/) • ESP PREDICTOR: not good predictions • CNB APPLICATION FOR HPP PROJECT: provided by A. Medina. • EQUIPMENT: • AB SCIEX QTRAP 5500. • QTRAP RUNNING CONDITIONS: 60 min. gradient. • Protocol: 1 protein per running in the analyst method. • PROCESSING SOFTWARES: • SKYLINE (v. 1.2.0.3425 MacCoss Lab Software) (https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view) • MRM PILOT (AB SCIEX 2.1.673.0 version) • ANALYST (AB SCIEX 1.6. version)

8. PROTEIN MRM PILOT/ANALYST ACQUISITION PARAMETERS

9. PROCESSING RESULTS SOFTWARE: SKYLINE WHY SKYLINE? • INTUITIVE • IT ALLOWS TO: • SEARCH UNIQUE PEPTIDES IN THE BACKGROUND PROTEOME (FASTA UNIPROT/SP TAX. HUMAN). • COMPARE OUR RESULTS WITH OTHER SPECTRA DATABASES. • INTEGRATE PEAKS AND COMPARE SEVERAL RUNNINGS. • EXPORT TO DATASHEETS. • CREATE ACQUISITIONS METHODS. • SHARE FILES AND IMPORT FROM DIFFERENT DATA FORMATS. • IT IS FREE

10. RESULTS MRM DISCOVERY FOR THE PROTEINS AND PEPTIDES OF THE ENTIRE 3 EXTRACTS CELL LINES

11. POSSIBLE (but not confirmed) PEPTIDES IN SAMPLES

12. CONCLUSIONS • QTRAP IS A VERY SPECIFIC TOOL TO FIND THE PROTEINS OF INTEREST • GOOD ELECTION TO DEFINE THE ACQUSITION METHOD. • PROBLEMS WITH THE SENSIBILITY DUE TO THE LOW PROTEIN CONCENTRATION AND THE CHROMATOGRAPHIC SEPARATION. • POSSIBILITIES TO FIND ALL PROTEINS. SYNTHETIC PEPTIDES. • IT’S NECCESSARY TO OPTIMIZE PARAMETERS. • THE NORMALIZATION OF THE METHOD WITH ALL THE TEAMS IS AN ESSENTIAL ITEM.

13. THE NOMO1 CASE

15. 3 Genes match yourquery ('NOMO1') in Human NOMO1 Description NODAL modulator 1 [Source:HGNC Symbol;Acc:30060] [Type: proteincodingEnsembl/Havanamerge genes] Gene ID ENSG00000103512 Location 16:14927538-14990017:1 NOMO3 Description NODAL modulator 3 [Source:HGNC Symbol;Acc:25242] [Type: proteincodingEnsembl/Havanamerge genes] Gene ID ENSG00000103226 Location 16:16326352-16388668:1 NOMO2 Description NODAL modulator 2 [Source:HGNC Symbol;Acc:22652] [Type: proteincodingEnsembl/Havanamerge genes] Gene ID ENSG00000185164 Location 16:18511182-18573533:-1

19. NOMO1 PROTEIN • ENSEMBL: 3 GENES IN CHROMOSOME 16 WITH DIFFERENT LOCATIONS. THEY ARE NOT THE SAME PROTEIN WITH DIFFERENT ISOFORMS. • ONLY A PROTEOTYPIC PEPTIDE BETWEEN THE 3: ALGQAASDNSGPEDAK [1195, 1210]. • IT IS ESSENTIAL TO FIND 3 UNIQUE PEPTIDES TO CONFIRM THE EXISTENCE OF NOMO1. • CHECKING SOME LITERATURE ONLY THE MIXTURE OF NOMO PROTEINS WERE FOUND.

20. WHAT MUST WE DO?

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