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PRELIMINARY RESULTS OF THE MRM ANALYSIS

PRELIMINARY RESULTS OF THE MRM ANALYSIS. sHPP CHROMOSOME 16 MEETING August, 28, 2012 La Cristalera, Miraflores de la Sierra. INDEX. ASSIGNED PROTEINS AND PROBLEMS MATERIALS AND METHODS RESULTS: ENTIRE EXTRACTS OF THE 3 CELL LINES SAMPLES CONCLUSIONS THE NOMO1 CASE.

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PRELIMINARY RESULTS OF THE MRM ANALYSIS

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  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

  14. 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

  15. 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.

  16. WHAT MUST WE DO?

  17. THANK YOU

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