Les contes des mille et une protéines... Thermococcus gammatolerans, une archée d abysse californienne
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1 Les contes des mille et une protéines... Deux organismes résistants à des dommages massifs de l ADN: Deinococcus deserti, une bactérie isolée du Sahara & Electron microscopy of D. deserti cells Thermococcus gammatolerans, une archée d abysse californienne Electron microscopy of T. gammatolerans cell Jean Armengaud Institut Biologie Environnementale & Biotechnologie Bagnols-sur-Cèze
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3 Chromosome 3 MPlasmids y n a m ow es? H n ge pb pb pb pb pb 3051 CDS? 3051 proteins? GenOMICS ProteOMICS +trnas/rrnas PTMs?
4 A Reference map of D. deserti proteome Identification of 140 proteins
5 Proteogenomic annotation: the very-large shotgun approach Cells production Proteome fractioning Trypsin nanolc-ms/ms MS/MS spectrum Databases Mascot (v2.2.01) search IRMA (v1.11.1) parsing Peptide assignment Matching peptides and nucleic acid sequences
6 The «One Thousand and One proteins» tale MS/MS spectra ( E O) spectra were assigned 369 nanolc-ms/ms (326 E + 40 O) unique peptides proteins (0.2% FP) Most abundant proteins (40% of the theoretical proteome [3457])
7 Mining the proteome from top of the iceberg to bottom spectra were assigned proteins?
8 When proteomics helps genomics Automatic annotation Proteomic data Validation A new integrated software for annotation Philippe Ortet & Mohamed Barakat (ibeb-sbvme-lemire) 250 hypothetical conserved proteins were confirmed 48 orphans were confirmed
9 The Sahara and some mirages. Chromosome region view Frames C_ _ Deide19965 MLKTPEIRRVRLFDLVPRDPASGLIDIPGGDLMQELACTWVPGTLDIVRLKVGT STIELTSTRLARIFGPQALNDLYLKGRAVVKADARQVAMLA A really new protein! No known homolog found by means of Psi-BLAST for this 95 residues protein C_ _ Cyan => peptide with score <40 Black => peptide with score >40 Deide19972 MTDNHEQRAQTEQTPEELRDIIPQLQGEPDEDPVLDAEEADTEAGTDASAAV GAEDDGEELEDEFIDADDLLALLSEMKEMLEAQGKEIRGLRREMREMREAQG QGGGFRGGDRSSGGDRGPSGGDRQGGGGFRPREDRGGFQDRSGGGDRG GYRGGGDRGFSGGDRQGGGGFRPREDRGGDRGFGGGDRQGGGGFRPRE DRGGFQDRSGGGDRGGYRGGGDRGFGGGDRQGGGGFRPREDRGGFQDR SGGGDRGGFRPRDDRGDRPAPQSGGFQDREFRPRDTDAGAGDGGFRPRA RADRGWGNKRTDEE The gene is on the reverse strand! Homologues exclusively found in Deinococci 15 new genes and 11 new oriented genes
10 N-terminal peptides 201 peptidic signatures 136 N-ter 112 proteins 24 N-ter corrected >Deide23190 MVDPMMSEAHTTPVEPTAPPRGLLIVMTGASGVGKGTLRELWLRDQDVFYSTSWTTREARPGEVDGVDYIFVSADAFEQKV QQGGFLEHASFVGNHYGTPVEPIEAALSRGQDVILEIEVEGAMQVRDRMGDEAILVFIMPPSLTELRRRLEGRATETPERI EKRLARAREEIMHAHAFRYVIVNDDLNRAVQELEAVQGAERARQRPESSWTPEDRAAVERAAQVRSDALSEADLLHVVNS First initiation codon Second initiation codon MMSEAHTTPVEPTAPPR MSEAHTTPVEPTAPPR Methionine removed SEAHTTPVEPTAPPR
11 How to go further with protein N-termini? 112 proteins 24 N-ter corrected (1/5!) (8% of 1348 proteins detected) chemical modification! trypsin + protein purification High accuracy nanolc-ms/ms LTQ-Orbitrap MS/MS spectrum of [TMPP + -Ac-ASNK-OH] 2+ at m/z
12 Purification of nucleoïds from Deinococci purification trypsin Shotgun analysis Free-label quantification Optimization of several purification steps 205 proteins quantified (700 proteins detected) 4 differents samples (from the same protocole) 2 different measurements
13 Thermococcus gammatolerans DNA repair system Shotgun analysis General metabolism 2.0 Mbp 114 nanolc-ms/ms MS/MS spectra assignments (40%) unique peptides p<0.001 (score ionique >25) proteins (0.5% FP) (50% of the theoretical proteome [2157]) 155 protein N-termini (22 acetylated) 20 N-ter corrected
14 Many thanks to... Elodie Sahinovic Mathieu Baudet Philippe Guérin Bernard Fernandez Alain Dedieu Jean Armengaud ibeb-sbtn-lbsp (Marcoule) But also Charles, Véronique, Jean-Charles, and for your kind attention! Suzanne Sommer Magali Toueille IGM (Orsay) Arjan de Groot Rémi Dulermo Laurence Blanchard Mohamed Barakat Philippe Ortet Wafa Achouak Thierry Heulin ibeb-sbvme-lemire (Cadarache) Yvan Zivanovic Arnaud Lagorce Fabrice Confalonieri IGM (Orsay) Projet Blanc: Deinococcus
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