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

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Presentation transcript:

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

KNOWN PROTEINS ASSIGNED TO THE UCM- PCM PROTEOMICS UNIT ENSEMBLE ID ACCESSION NUMBERGENE IDPROTEIN ID ENSG P60827C1QT8_HUMANC1QTNF8 ENSG P57073SOX8_HUMANSOX8 ENSG Q02223TNR17_HUMANTNFRSF17 ENSG Q96J65MRP9_HUMANABCC12 ENSG Q08AH1ACSM1_HUMANACSM1 ENSG Q8WYQ9ZCH14_HUMANZCCHC14

MATERIALS AND METHODS

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.

PROTEINS AND PEPTIDES SEARCH PROTEIN SEQUENCES USING SWISSPROT ACCESSION NUMBER. ( ENSEMBL ENTRANCES : ( NCBI BLAST TO SEARCH PROTEOTYPIC PEPTIDES. ( 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.

EQUIPMENTS AND SOFTWARES PREDICTOR SOFTWARES: –SRM ATLAS ( –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 MacCoss Lab Software) ( –MRM PILOT (AB SCIEX version) –ANALYST (AB SCIEX version)

PROTEIN MRM PILOT/ANALYST ACQUISITION PARAMETERS PROTEIN IDTRANSITIONSPEPTIDESDWELL TIMEOBSERVATIONS C1QTNF res. 1 miss, M, variable mod. SOX res. 1 miss TNFRSF res. 1 miss, M, variable mod. ABCC res. 0 miss ACSM res. 0 miss ZCCHC res. 0 miss

MCF7 CELLS RESULTS OFF-GEL FRACTIONS

MCF-7 10% ACRYLAMIDE SDS PAGE SAMPLE MW IEF FRACTIONS MCF7 SAMPLE

RAMOS CELL LINE RESULTS MRM DISCOVERY

POSSIBLE (but not confirmed) PEPTIDES IN SAMPLES PROTEIN ID Nº ANALYST METHOD PEPTIDES 3 OR MORE TRANS. PEPTIDES 2 TRANS. PEPTIDES C1QTNF SOX8 710 TNFRSF ABCC ACSM ZCCHC

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.