1. HPP Preliminary Results La Cristalera, 28-29 August 2012 Montserrat Carrascal, Joan Villanueva, Joaquín Abián LP-CSIC/UAB

2. M. Carrascal 2011 Protein share out : LP-CSIC/UAB TARGET PEPTIDE SELECTION:   Peptide Siever: list of potential peptides.   Cys, Met and missed cleavage are eliminated.   Other scores are calculated: STEPP and PeptideDetectabilityPredictor.   Average of Peptide Siever, STEPP and PeptideDetectabilityPredictor. Filtering at 0.75 or 4 peptides/protein. M.Carrascal 2012

3. M. Carrascal 2011 TARGET IONS M.Carrascal 2012

4. M. Carrascal 2011 SAMPLE PREPARATION: Half of the MCF-7 and CDC-18 samples (aprox. 250 µg protein) were fractionated using the Offgel system (Agilent).12 fractions were obtained and digested with FASP. INSTRUMENTAL ANALYSIS: The resulting extracts were injected in 2 different LC/MS systems ANALYSIS VELOS: 1/6 of each extract was injected. HPLC gradient 120 min MS programming: 1 MS followed by 30 Targeted MS/MS ANALYSIS ORBITRAP: 1/6 of each extract was injected. HPLC gradient 120 min MS programming: 1 MS followed by 10 Data Dependent MS/MS from inclusion list MS were obtained with FT at resolution 100000, MS/MS were obtained with IT EXPERIMENTAL DESIGN M.Carrascal 2012

5. M. Carrascal 2011 TARGETED MS/MS : LTQ VELOS Sample VH: MCF-7 MS traces Offgel Fr6 Offgel Fr7 M.Carrascal 2012

6. M. Carrascal 2011 DATA DEPENDENT WITH INCLUSION LIST: LTQ-ORBITRAP Offgel Fr6 Offgel Fr7 Sample VH: MCF-7 MS traces M.Carrascal 2012

7. M. Carrascal 2011 ANALYSIS VELOS: 1.- The resulting RAW files were searched using Proteome Discoverer v1.3 against a database including only the 6 targeted proteins. Precursor tolerance 2Da. Product tolerance 0.8Da 2.- Matches with a Xcorr 1.8 were checked manually. DATA ANALYSIS ANALYSIS ORBITRAP: the RAW data was processed twice: METHOD ORBITRAP 1: The RAW data was searched against a database with 6 proteins (MS tolerance of 10ppm). RESULT METHOD ORBITRAP 1: No hits were returned by the Proteome Discoverer software. METHOD ORBITRAP 2: The targeted MS data was revised manually : 1.- Fragmentograms for each proteotypic peptide were obtained at z=1, 2, 3 and 4. To ensure selectivity resolution was reduced to 10 ppm 2.- Intense LCMS peaks or small peaks observed in two of the 4 fragmentograms were manually checked: - have the expected z values - Is not related to isotopic contamination of other peptides M.Carrascal 2012

8. M. Carrascal 2011 CHROMOSOME 16 PROTEIN DESCRIPTION Target peptides undetected OffGel (12 fractions) FASP digestion LC-MS/MS Protein Discovery Manual inspection Low amount of target proteins Proteins not expressed in these cells ensembl_gene_idexternal_gene_idEntryProtein.existence ENSG00000179583CIITAP33076Evidence at protein level ensembl_gene_idexternal_gene_idatlas_celltype ENSG00000179583CIITACD56+ NK cell M.Carrascal 2012

9. M. Carrascal 2011 CHROMOSOME 16 PROTEIN DESCRIPTION UNIPROT 2012_07 Chrom16.txt M.Carrascal 2012

10. M. Carrascal 2011 CHROMOSOME 16 PROTEIN DESCRIPTION Peptides and proteins from T-CELLS in chromosome 16 Peptides and proteins from PLASMA in chromosome 16 M.Carrascal 2012  Review of other studies (different cells or tissues).  Creation of a database of spectra for MRM experiments.

11. M. Carrascal 2011 M.Carrascal 2012 SEProt 2013 AND HPP

12. M. Carrascal 2011 M.Carrascal 2012