title: N-terminome analysis of the human mitochondrial proteome.
abstract: The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Alvaro Sebastian
authors.initials: AS
authors.lastname: Vaca Jacome
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UGA 5249, iRTSV/LCBM, CEA Grenoble, Grenoble, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Rabilloud
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Schaeffer-Reiss
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Rompais
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ayoub
authors.affiliation: CALIPHO Group, SIB-Swiss Institute of Bioinformatics, Geneva, Switzerland.|Department of Human Protein Sciences, Faculty of Medicine, Geneva, Switzerland.
authors.forename: Lydie
authors.initials: L
authors.lastname: Lane
authors.affiliation: CALIPHO Group, SIB-Swiss Institute of Bioinformatics, Geneva, Switzerland.|Department of Human Protein Sciences, Faculty of Medicine, Geneva, Switzerland.
authors.forename: Amos
authors.initials: A
authors.lastname: Bairoch
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), Université de Strasbourg, IPHC, Strasbourg, France.|IPHC, CNRS, UMR7178, Strasbourg, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Carapito
mesh_terms: D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D024101:Mitochondrial Proteins / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods*
chemical_list: D024101:Mitochondrial Proteins; D009943:Organophosphorus Compounds; C405845:tris(trimethoxyphenyl)phosphonium acetyl
keywords: Animal proteomics; Free N-terminome analysis; Human mitochondria; Proteogenomics; Transit peptide; dN-TOP approach