Post on 09-Jun-2020
transcript
Les contes des mille et une protéines...
Jean Armengaud
Deux organismes résistants à des dommages massifs de l’ADN:
Deinococcus deserti, une bactérie isolée du Sahara
Thermococcus gammatolerans, une archée d’abysse californienne
Electron microscopyof D. deserti cells
Electron microscopyof T. gammatolerans cell
&
Institut Biologie Environnementale & BiotechnologieBagnols-sur-Cèze
GenOMICS
2 819 842 pb
324 711 pb
314 317 pb
396 459 pb
3 855 329 pb
3051 CDS?
3 MPlasmidsChromosome
+tRNAs/rRNAs…How many
genes? ProteOMICS
3051 proteins?
PTMs?
A Reference map of D. deserti proteome
Identification of 140 proteins
Proteogenomic annotation: the very-large shotgun approach
Databases
MS/MS spectrum Peptide assignmentMascot (v2.2.01) search
IRMA (v1.11.1) parsing
98
49
10
28
Proteome fractioning Trypsin nanoLC-MS/MSCells production
+
Matching peptides and nucleic acid sequences
369 nanoLC-MS/MS
264 251 MS/MS spectra
11 129 unique peptides
50 669 spectra were assigned
(40% of the theoretical proteome [3457])
The « One Thousand and One proteins » tale…
1 348 proteins
Most abundant proteins…
(326 E + 40 O)
(163779 E + 100472 O)
(0.2% FP)
Mining the proteome……from top of the iceberg to bottom
50 669 spectra were assigned
1 348 proteins
?
Automatic annotation
Proteomic data
Validation
250 hypothetical conserved proteins were confirmed
48 orphans were confirmedPhilippe Ortet & Mohamed Barakat
(iBEB-SBVME-LEMIRE)
When proteomics helps genomics…
A new integratedsoftware for annotation
Chromosome region view
The Sahara and some mirages ….
+1+2+3
-1-2-3
Frames
C_2400239_-174868 1 79 71 12
111435 1 48 86 13
MLKTPEIRRVRLFDLVPRDPASGLIDIPGGDLMQELACTWVPGTLDIVRLKVGTSTIELTSTRLARIFGPQALNDLYLKGRAVVKADARQVAMLA
Deide19965
A really new protein!No known homolog found by means of Psi-BLAST for this 95 residues protein
Cyan => peptide with score <40Black => peptide with score >40
C_2403561_-334388 1 52 128 1080132 1 61 36 1194813 1 37 310 1432591 1 40 291 14
MTDNHEQRAQTEQTPEELRDIIPQLQGEPDEDPVLDAEEADTEAGTDASAAVGAEDDGEELEDEFIDADDLLALLSEMKEMLEAQGKEIRGLRREMREMREAQGQGGGFRGGDRSSGGDRGPSGGDRQGGGGFRPREDRGGFQDRSGGGDRGGYRGGGDRGFSGGDRQGGGGFRPREDRGGDRGFGGGDRQGGGGFRPREDRGGFQDRSGGGDRGGYRGGGDRGFGGGDRQGGGGFRPREDRGGFQDRSGGGDRGGFRPRDDRGDRPAPQSGGFQDREFRPRDTDAGAGDGGFRPRARADRGWGNKRTDEE
Deide19972
The gene is on the reverse strand!Homologues exclusively found in Deinococci
15 new genes and11 new oriented genes
>Deide23190 MVDPMMSEAHTTPVEPTAPPRGLLIVMTGASGVGKGTLRELWLRDQDVFYSTSWTTREARPGEVDGVDYIFVSADAFEQKVQQGGFLEHASFVGNHYGTPVEPIEAALSRGQDVILEIEVEGAMQVRDRMGDEAILVFIMPPSLTELRRRLEGRATETPERIEKRLARAREEIMHAHAFRYVIVNDDLNRAVQELEAVQGAERARQRPESSWTPEDRAAVERAAQVRSDALSEADLLHVVNS
N-terminal peptides
201 peptidic signatures 136 N-ter 112 proteins
24 N-ter corrected
MMSEAHTTPVEPTAPPR
MSEAHTTPVEPTAPPR
SEAHTTPVEPTAPPR
First initiation codon
Second initiation codon
Methionine removed
How to go further with protein N-termini ?
112 proteins 24 N-ter corrected (1/5 !)
(8% of 1348 proteins detected)
+ protein
trypsin
purification High accuracynanoLC-MS/MS
…chemical modification!
LTQ-Orbitrap MS/MS spectrum of [TMPP+-Ac-ASNK-OH] at m/z 496.212+
Purification of nucleoïds from Deinococci
Shotgun analysisFree-label quantification
4 differents samples(from the same protocole)
2 different measurements
purification trypsin
(700 proteins detected)205 proteins quantifiedOptimization
of severalpurification steps
Thermococcus gammatolerans…
Shotgun analysis
114 nanoLC-MS/MS442 916 MS/MS spectra167 497 assignments (40%)
10841 unique peptides
DNA repair system
General metabolism
(50% of the theoretical proteome [2157])
1 076 proteins (0.5% FP)
p<0.001 (score ionique >25)
155 protein N-termini
20 N-ter corrected
(22 acetylated)
2.0 Mbp
Many thanks to...Suzanne SommerMagali Toueille
IGM(Orsay)
Arjan de GrootRémi Dulermo
Laurence BlanchardMohamed BarakatPhilippe OrtetWafa AchouakThierry Heulin
iBEB-SBVME-LEMiRE(Cadarache)
Projet Blanc: Deinococcus
But also… Charles, Véronique, Jean-Charles,
… and for your kind attention !
Elodie SahinovicMathieu BaudetPhilippe Guérin
Bernard FernandezAlain Dedieu
Jean Armengaud
iBEB-SBTN-LBSP(Marcoule)
Yvan ZivanovicArnaud Lagorce
Fabrice ConfalonieriIGM(Orsay)