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HPP Preliminary Results La Cristalera , 28-29 August 2012

HPP Preliminary Results La Cristalera , 28-29 August 2012. Montserrat Carrascal, Joan Villanueva, Joaquín Abián LP-CSIC/UAB. Protein share out : LP-CSIC/UAB. TARGET PEPTIDE SELECTION: Peptide Siever : list of potential peptides . Cys , Met and missed cleavage are eliminated .

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HPP Preliminary Results La Cristalera , 28-29 August 2012

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  1. HPPPreliminary ResultsLa Cristalera, 28-29 August 2012 Montserrat Carrascal, Joan Villanueva, Joaquín Abián LP-CSIC/UAB

  2. Protein share out : LP-CSIC/UAB • TARGET PEPTIDE SELECTION: • PeptideSiever: list of potentialpeptides. • Cys, Met and missedcleavage are eliminated. • Other scores are calculated: STEPP and PeptideDetectabilityPredictor. • Average of PeptideSiever, STEPP and PeptideDetectabilityPredictor. Filtering at 0.75 or 4 peptides/protein. M.Carrascal 2012

  3. TARGET IONS M.Carrascal 2012

  4. EXPERIMENTAL DESIGN SAMPLE PREPARATION: Half of the MCF-7 and CDC-18 samples (aprox. 250 µg protein) werefractionatedusingtheOffgelsystem (Agilent).12 fractionswereobtained and digestedwith FASP. INSTRUMENTAL ANALYSIS: Theresultingextractswereinjected in 2 different LC/MS systems ANALYSIS VELOS: 1/6 of eachextractwasinjected. HPLC gradient 120 min MS programming: 1 MS followedby 30 Targeted MS/MS ANALYSIS ORBITRAP: 1/6 of eachextractwasinjected. HPLC gradient 120 min MS programming: 1 MS followedby 10 Data Dependent MS/MS frominclusionlist MS wereobtainedwith FT at resolution 100000, MS/MS wereobtainedwith IT M.Carrascal 2012

  5. TARGETED MS/MS : LTQ VELOS Sample VH: MCF-7 MS traces Offgel Fr6 Offgel Fr7 M.Carrascal 2012

  6. DATA DEPENDENT WITH INCLUSION LIST: LTQ-ORBITRAP Sample VH: MCF-7 MS traces Offgel Fr6 Offgel Fr7 M.Carrascal 2012

  7. DATA ANALYSIS ANALYSIS VELOS: 1.- Theresulting RAW files weresearchedusingProteomeDiscoverer v1.3 against a databaseincludingonlythe 6 targetedproteins. Precursor tolerance 2Da. Producttolerance 0.8Da 2.- Matcheswith a Xcorr < 1.8 werediscarded. MatcheswithXcorr>1.8 werecheckedmanually. ANALYSIS ORBITRAP: the RAW data wasprocessedtwice: METHOD ORBITRAP 1: The RAW data wassearchedagainst a databasewith 6 proteins (MS tolerance of 10ppm). RESULT METHOD ORBITRAP 1: No hits werereturnedbytheProteomeDiscoverer software. METHOD ORBITRAP 2: Thetargeted MS data wasrevisedmanually : 1.- Fragmentograms foreach proteotypic peptidewereobtained at z=1, 2, 3 and 4. Toensureselectivityresolutionwasreducedto 10 ppm 2.- Intense LCMS peaksorsmallpeaksobserved in two of the 4 fragmentograms weremanuallychecked: - havetheexpected z values - Isnotrelatedtoisotopiccontamination of otherpeptides M.Carrascal 2012

  8. CHROMOSOME 16 PROTEIN DESCRIPTION OffGel (12 fractions) FASP digestion LC-MS/MS ProteinDiscovery Manual inspection Target peptidesundetected • Lowamount of target proteins • Proteinsnotexpressed in thesecells M.Carrascal 2012

  9. CHROMOSOME 16 PROTEIN DESCRIPTION UNIPROT 2012_07 Chrom16.txt M.Carrascal 2012

  10. CHROMOSOME 16 PROTEIN DESCRIPTION Peptides and proteinsfrom T-CELLS in chromosome 16 Peptides and proteinsfrom PLASMA in chromosome 16 Review of other studies (different cells or tissues). Creation of a database of spectra for MRM experiments. M.Carrascal 2012

  11. SEProt 2013 AND HPP M.Carrascal 2012

  12. M.Carrascal 2012

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