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 ENSEMBLE ID ACCESSION NUMBERGENE IDPROTEIN ID ENSG00000184471P60827C1QT8_HUMANC1QTNF8 ENSG00000005513P57073SOX8_HUMANSOX8 ENSG00000048462Q02223TNR17_HUMANTNFRSF17 ENSG00000140798Q96J65MRP9_HUMANABCC12 ENSG00000166743Q08AH1ACSM1_HUMANACSM1 ENSG00000140948Q8WYQ9ZCH14_HUMANZCCHC14

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/)http://www.uniprot.org/ ENSEMBL ENTRANCES : (http://www.ensembl.org/index.html)http://www.ensembl.org/index.html NCBI BLAST TO SEARCH PROTEOTYPIC PEPTIDES. (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)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/)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)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 PROTEIN IDTRANSITIONSPEPTIDESDWELL TIMEOBSERVATIONS C1QTNF8705157-19 res. 1 miss, M, variable mod. SOX8727208-15 res. 1 miss TNFRSF17594207-19 res. 1 miss, M, variable mod. ABCC129512108-15 res. 0 miss ACSM19811107-19 res. 0 miss ZCCHC1411211108-15 res. 0 miss

9. MCF7 CELLS RESULTS OFF-GEL FRACTIONS

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

11. RAMOS CELL LINE RESULTS MRM DISCOVERY

12. POSSIBLE (but not confirmed) PEPTIDES IN SAMPLES PROTEIN ID Nº ANALYST METHOD PEPTIDES 3 OR MORE TRANS. PEPTIDES 2 TRANS. PEPTIDES C1QTNF8 5 1 0 SOX8 710 TNFRSF17 411 ABCC12 1000 ACSM1 1103 ZCCHC14 1110

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.