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HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP

HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28 th and 29 th August 2012. Introduction. CIC bioGUNE 6 unknown proteins. Ramos cells ( lymphoma ). MCF7 cells ( breast cancer ). CCD18 cells (colon fibroblast ).

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HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP

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  1. HPP ch16 CIC bioGUNE RESULTS MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  2. Introduction CIC bioGUNE 6 unknownproteins Ramos cells (lymphoma) MCF7 cells (breastcancer) CCD18 cells (colon fibroblast) ·700ug/25ul (28ug/ul aprox.) ·500 ug/ 50ul (10ug/ul) ·500 ug/ 700ul (0.7ug/ul) MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  3. Gels MCF-7 RAMOS & CCD18 MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  4. Workflow Trypsin ANALYSIS by LC-MS MCF7 (50ug) Ramos (10ug) Resuspension in FA0.1% MS LC Thermo LTQ-Orbitrap XL ETD WatersnanoAcquity Symmetry 300 C18 UPLC trap (180 µm x 20 mm, 5 µm) BEH130 C18 column (75 μm x 200 mm, 1.7 μm) 60 min. linear gradient 3%-40% B (ACN FA0.1%) DATABASES SEARCH ENGINES MASCOT SEQUEST ch16 HUMAN HPP cic MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  5. Results MCF7: MASCOT searches Exclusionlists 1st: DDA 2nd: Excllists MCF7 1-17 excMCF7 1-17 HUMAN ch16 HPP cic MASCOT Decoy (FDR<1%) MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  6. Results MCF7: SEQUEST searches Exclusionlists 1st: DDA 2nd: Excllists MCF7 1-17 excMCF7 1-17 HPP SEQUEST No Decoy ¿? MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  7. Results MCF7: NPW_HUMAN AAGLLMGLR SISGPISK (Lanosterolsynthase) Vs. ch16 Scanevent 1035 Scanevent 1035 DLTSPEPAAR DKFSFDLGK (Peptidyl-prolylcis-transisomerase) Vs. ch16 Scanevent 4351 Scanevent 4351 Inclusionlists – NO HPP HIT MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  8. Results RAMOS: MASCOT searches 1st: DDA RAMOS 1-23 HUMAN ch16 HPP cic MASCOT Decoy (FDR<1%) MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  9. Results: HPP target+decoysearches Mascot No decoy (p<0.05) MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  10. Summary 1-Protein quantissue 2-No HPP proteinsidentified: *Only NPW_HUMAN wasidentified – false positive *Differentapproaches? Inclusionlists???... MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  11. NPW_HUMAN Swissprot TrEMBL

  12. Results (7): MTHSFD_HUMAN MEPRAGVSKQDIR

  13. Mann 2012 Virtual digestion Workflow (2): MCF7 cells ch16 HUMAN HPP7 MASCOT SEQUEST SEARCH ENGINES DATABASES Inclusionlists vs. ch16 DB 1st: DDA S MCF7 1-17 M Exclusionlists 2nd: Excllists vs. HUMAN vs. ch16 DB S excMCF7 1-17 M Acquisition Confirmation vs. HUMAN 3rd: Incllists incMCF7 S M S M vs. HUMAN vs. ch16 DB

  14. Results: Exclusionlists Vs. Chr16 Vs. HUMAN Exclist DDA Exclist DDA NO HACER COMPARATIVA, NO MERECE LA PENA, SOLO CITAR NUMEROS TOTALES CH16 Y HUMAN

  15. MCF7 cells MCF7 cells ·500 ug/ 50ul (10ug/ul) CIC bioGUNE 6 unknownproteins MEETING OF THE SPANISH CONSORTIUM FOR THE PARTICIPACION IN THE HPP La Cristalera, 28th and 29thAugust 2012

  16. Results (1): MASCOT searches Highconfidence hits (FDR<1%) Mascot vs ch16 & HUMAN DB. HPP hits = 0

  17. Workflow (2) MCF7 1 - 17 Incllists Vs. HPP DB 1st ACQUISITION Survey (30000 FWHM) DDA CID 6 most intense MS/MS trigger1000counts Vs. Ch16 DB Vs. Human DB Excllists 2nd ACQUISITION: Exclusionlists Survey (30000 FWHM) DDA CID 6 most intense. MS/MS trigger400counts 3rd ACQUISITION: Inclusionlists Survey (30000 FWHM) Inclusionlist MS/MS trigger400counts

  18. Searches & Peptidelists SequestRelaxedsearches ·No decoy ·High & Mediumconfidence hits ·Define inclusionlists Mascotstringentsearches ·Decoy ·Highconfidence hits (FDR<1%) ·Define proteinIds & exclusionlists

  19. Mann 2012 Virtual digestion Workflow (3): HPP7 DB ch16 HUMAN HPP7 SEQUEST MASCOT Inclusionlists vs. HPP7 DB 1st: DDA S MCF7 1-17 M Exclusionlists 2nd: Excllists vs. HUMAN vs. HPP7 DB S excMCF7 1-17 M Acquisition Confirmation vs. HUMAN 3rd: Incllists incMCF7 S M S M vs. HUMAN vs. ch16 DB

  20. Results (2): SEQUEST searches High, med and lowconfidence hits (no decoy) vs. HPP7 DB

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