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FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS

FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS. sHPP CHROMOSOME 16 MEETING August, 28, 2012 La Cristalera, Miraflores de la Sierra . INDEX. ASSIGNED PROTEINS MATERIALS AND METHODS RESULTS: MCF7 OFF GEL FRACTIONS RAMOS CELL LINE ENTIRE EXTRACT RESULTS CONCLUSIONS AND FUTURE PERSPECTIVES.

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FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS

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  1. FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS sHPP CHROMOSOME 16 MEETING August, 28, 2012 La Cristalera, Miraflores de la Sierra

  2. INDEX • ASSIGNED PROTEINS • MATERIALS AND METHODS • RESULTS: • MCF7 OFF GEL FRACTIONS • RAMOS CELL LINE ENTIRE EXTRACT RESULTS • CONCLUSIONS AND FUTURE PERSPECTIVES

  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) • OFF-GEL SAMPLES: • ONLY FOR MCF-7 CELL LINE. • 12 fractions, following the provided protocol. • IN SOLUTION DIGESTIONS OF THE PROTEIN EXTRACT: • Usual own lab protocol. • Digestion for the entire extracts 3 lines. Only Ramos cells were used to search our unknown proteins.

  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 TO SEARCH PROTEOTYPIC PEPTIDES. (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) • MATCHING OF PEPTIDES FOR ACQUISITION METHOD: • GPM AND NIST SPECTRA DATABASES FOR SKYLINE SOFTWARE. NO RESULTS WERE FOUND. • SKYLINE DIGESTION AND UNIQUE PEPTIDES SEARCH USING THE BACKGROUND PROTEOME. • MRM PILOT.

  7. EQUIPMENTS AND SOFTWARES • PREDICTOR SOFTWARES: • SRM ATLAS(http://www.srmatlas.org/) • ESP PREDICTOR: not good predictions • EQUIPMENT: • AB SCIEX QTRAP 5500 (CNB) • QTRAP RUNNING CONDITIONS: 60 min. gradient. • Protocol: 1 protein per running in the analyst method. • Agilent Technologies 3100 Offgel Fractionator. • Protocol: WE FOLLOWED THE PROVIDED PROTOCOL. • 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 1.6.0 version)

  8. PROTEIN MRM PILOT/ANALYST ACQUISITION PARAMETERS

  9. MCF7 CELLS RESULTS OFF-GEL FRACTIONS

  10. MCF-7 10% ACRYLAMIDE SDS PAGE SAMPLE MW MCF7 SAMPLE MW IEF FRACTIONS 250 150 100 75 50 37 25 15 10

  11. RAMOS CELL LINE RESULTS MRM DISCOVERY

  12. POSSIBLE (but not confirmed) PEPTIDES IN SAMPLES

  13. CONCLUSIONS AND FUTURE PERSPECTIVES • OFF GEL: BAD PROTEIN SEPARATION. • QTRAP DIDN´T WORK FORTHE ENTIRE RAMOS EXTRACT. • PREVIOUS SEPARATION OF THE SAMPLE . • CARRY OUT PREVIOUS SHOTGUN WITH OFF GEL FRACTIONS. • CHECK QTRAP WITH THE OFFGEL MCF7 FRACTIONS. • OPTIMIZE PARAMETERS AND TO SOLVE PROBLEMS. • SYNTHETIC PEPTIDES. • NORMALIZATION OF THE METHODS.

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