title: Computational and Mass-Spectrometry-Based Workflow for the Discovery and Validation of Missing Human Proteins: Application to Chromosomes 2 and 14.
abstract: In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last 4 years, the French proteomics infrastructure has collected high-quality data sets from 40 human samples, including a series of rarely studied cell lines, tissue types, and sample preparations. Here we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these data sets. Screening database search results (85,326 dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-09-19). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level false discovery rate calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from four of the missing proteins detected in tissue/cell samples, which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for three of the proteins. For two of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178 , 25 Rue Becquerel, 67087 Strasbourg, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Carapito
authors.affiliation: CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU , rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.|Department of Human Protein Sciences, Faculty of Medicine, rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.
authors.forename: Lydie
authors.initials: L
authors.lastname: Lane
authors.affiliation: CEA, DSV, iRTSV, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble, F-38054, France.|INSERM U1038 , 17, rue des Martyrs, Grenoble F-38054, France.|Université Grenoble , Grenoble F-38054, France.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Benama
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178 , 25 Rue Becquerel, 67087 Strasbourg, France.
authors.forename: Alisson
authors.initials: A
authors.lastname: Opsomer
authors.affiliation: CNRS UMR5089 Institut de Pharmacologie et de Biologie Structurale, 118 route de Narbonne, 31077 Toulouse, France.|Université de Toulouse , 205, route de Narbonne, 31077 Toulouse, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Mouton-Barbosa
authors.affiliation: CNRS UMR5089 Institut de Pharmacologie et de Biologie Structurale, 118 route de Narbonne, 31077 Toulouse, France.|Université de Toulouse , 205, route de Narbonne, 31077 Toulouse, France.
authors.forename: Luc
authors.initials: L
authors.lastname: Garrigues
authors.affiliation: CNRS UMR5089 Institut de Pharmacologie et de Biologie Structurale, 118 route de Narbonne, 31077 Toulouse, France.|Université de Toulouse , 205, route de Narbonne, 31077 Toulouse, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Gonzalez de Peredo
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178 , 25 Rue Becquerel, 67087 Strasbourg, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Burel
authors.affiliation: CEA, DSV, iRTSV, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble, F-38054, France.|INSERM U1038 , 17, rue des Martyrs, Grenoble F-38054, France.|Université Grenoble , Grenoble F-38054, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bruley
authors.affiliation: CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU , rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.|Department of Human Protein Sciences, Faculty of Medicine, rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.
authors.forename: Alain
authors.initials: A
authors.lastname: Gateau
authors.affiliation: CNRS UMR5089 Institut de Pharmacologie et de Biologie Structurale, 118 route de Narbonne, 31077 Toulouse, France.|Université de Toulouse , 205, route de Narbonne, 31077 Toulouse, France.
authors.forename: David
authors.initials: D
authors.lastname: Bouyssié
authors.affiliation: CEA, DSV, iRTSV, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble, F-38054, France.|INSERM U1038 , 17, rue des Martyrs, Grenoble F-38054, France.|Université Grenoble , Grenoble F-38054, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Jaquinod
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178 , 25 Rue Becquerel, 67087 Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Cianferani
authors.affiliation: CNRS UMR5089 Institut de Pharmacologie et de Biologie Structurale, 118 route de Narbonne, 31077 Toulouse, France.|Université de Toulouse , 205, route de Narbonne, 31077 Toulouse, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178 , 25 Rue Becquerel, 67087 Strasbourg, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.affiliation: CEA, DSV, iRTSV, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble, F-38054, France.|INSERM U1038 , 17, rue des Martyrs, Grenoble F-38054, France.|Université Grenoble , Grenoble F-38054, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Garin
authors.affiliation: CEA, DSV, iRTSV, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble, F-38054, France.|INSERM U1038 , 17, rue des Martyrs, Grenoble F-38054, France.|Université Grenoble , Grenoble F-38054, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Vandenbrouck
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D002883:Chromosomes, Human, Pair 14; D002889:Chromosomes, Human, Pair 2; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011506:Proteins
keywords: Human Proteome Project (C-HPP); LC−SRM assays; bioinformatics; mass spectrometry; missing proteins identification