Les contes des mille et une protéines... Deinococcus deserti ...
Les contes des mille et une protéines... Deinococcus deserti ...
Les contes des mille et une protéines... Deinococcus deserti ...
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<strong>Les</strong> <strong>contes</strong> <strong>des</strong> <strong>mille</strong> <strong>et</strong> <strong>une</strong> <strong>protéines</strong>...<br />
Deux organismes résistants à <strong>des</strong> dommages massifs de l’ADN:<br />
<strong>Deinococcus</strong> <strong>des</strong>erti,<br />
<strong>une</strong> bactérie isolée du Sahara<br />
&<br />
Thermococcus gammatolerans,<br />
<strong>une</strong> archée d’abysse californienne<br />
Jean Armengaud<br />
Institut Biologie Environnementale & Biotechnologie<br />
Bagnols-sur-Cèze<br />
Electron microscopy<br />
of D. <strong>des</strong>erti cells<br />
Electron microscopy<br />
of T. gammatolerans cell
Chromosome<br />
2 819 842 pb<br />
324 711 pb<br />
314 317 pb<br />
396 459 pb<br />
3 855 329 pb<br />
How many<br />
genes?<br />
3 MPlasmids<br />
3051 CDS?<br />
GenOMICS<br />
+tRNAs/rRNAs…<br />
3051 proteins?<br />
ProteOMICS<br />
PTMs?
A Reference map of D. <strong>des</strong>erti proteome<br />
Identification of 140 proteins
Proteogenomic annotation: the very-large shotgun approach<br />
Cells production<br />
MS/MS spectrum<br />
+<br />
98<br />
49<br />
28<br />
10<br />
Proteome fractioning Trypsin nanoLC-MS/MS<br />
Databases<br />
Mascot (v2.2.01) search<br />
IRMA (v1.11.1) parsing<br />
Matching pepti<strong>des</strong> and nucleic acid sequences<br />
Peptide assignment
The « One Thousand and One proteins » tale…<br />
369 nanoLC-MS/MS<br />
(326 E + 40 O)<br />
Most abundant proteins…<br />
264 251 MS/MS spectra<br />
(163779 E + 100472 O)<br />
50 669 spectra were assigned<br />
11 129 unique pepti<strong>des</strong><br />
1 348 proteins<br />
(0.2% FP)<br />
(40% of the theor<strong>et</strong>ical proteome [3457])
Mining the proteome…<br />
…from top of the iceberg to bottom<br />
50 669 spectra<br />
were assigned<br />
1 348 proteins<br />
?
When proteomics helps genomics…<br />
Automatic annotation<br />
Proteomic data<br />
Validation<br />
A new integrated<br />
software for annotation<br />
Philippe Ort<strong>et</strong> & Mohamed Barakat<br />
(iBEB-SBVME-LEMIRE)<br />
250 hypoth<strong>et</strong>ical conserved<br />
proteins were confirmed<br />
48 orphans were confirmed
The Sahara and some mirages ….<br />
Frames<br />
+1<br />
+2<br />
+3<br />
-1<br />
-2<br />
-3<br />
C_2400239_-1<br />
74868 1 79 71 12<br />
111435 1 48 86 13<br />
Chromosome region view<br />
Deide19965<br />
MLKTPEIRRVRLFDLVPRDPASGLIDIPGGDLMQELACTWVPGTLDIVRLKVGT<br />
STIELTSTRLARIFGPQALNDLYLKGRAVVKADARQVAMLA<br />
A really new protein!<br />
No known homolog found by means of Psi-BLAST for this 95 residues protein<br />
C_2403561_-3<br />
34388 1 52 128 10<br />
80132 1 61 36 11<br />
94813 1 37 310 14<br />
32591 1 40 291 14<br />
Cyan => peptide with score peptide with score >40<br />
Deide19972<br />
MTDNHEQRAQTEQTPEELRDIIPQLQGEPDEDPVLDAEEADTEAGTDASAAV<br />
GAEDDGEELEDEFIDADDLLALLSEMKEMLEAQGKEIRGLRREMREMREAQG<br />
QGGGFRGGDRSSGGDRGPSGGDRQGGGGFRPREDRGGFQDRSGGGDRG<br />
GYRGGGDRGFSGGDRQGGGGFRPREDRGGDRGFGGGDRQGGGGFRPRE<br />
DRGGFQDRSGGGDRGGYRGGGDRGFGGGDRQGGGGFRPREDRGGFQDR<br />
SGGGDRGGFRPRDDRGDRPAPQSGGFQDREFRPRDTDAGAGDGGFRPRA<br />
RADRGWGNKRTDEE<br />
The gene is on the reverse strand!<br />
Homologues exclusively found in Deinococci<br />
15 new genes and<br />
11 new oriented genes
N-terminal pepti<strong>des</strong><br />
201 peptidic signatures 136 N-ter 112 proteins<br />
24 N-ter corrected<br />
>Deide23190<br />
MVDPMMSEAHTTPVEPTAPPRGLLIVMTGASGVGKGTLRELWLRDQDVFYSTSWTTREARPGEVDGVDYIFVSADAFEQKV<br />
QQGGFLEHASFVGNHYGTPVEPIEAALSRGQDVILEIEVEGAMQVRDRMGDEAILVFIMPPSLTELRRRLEGRATETPERI<br />
EKRLARAREEIMHAHAFRYVIVNDDLNRAVQELEAVQGAERARQRPESSWTPEDRAAVERAAQVRSDALSEADLLHVVNS<br />
First initiation codon<br />
Second initiation codon<br />
M<strong>et</strong>hionine removed<br />
MMSEAHTTPVEPTAPPR<br />
MSEAHTTPVEPTAPPR<br />
SEAHTTPVEPTAPPR
How to go further with protein N-termini ?<br />
112 proteins 24 N-ter corrected (1/5 !)<br />
(8% of 1348 proteins d<strong>et</strong>ected)<br />
+ protein<br />
…chemical modification!<br />
trypsin<br />
purification<br />
LTQ-Orbitrap MS/MS spectrum of [TMPP + -Ac-ASNK-OH]<br />
2+<br />
at m/z 496.21<br />
High accuracy<br />
nanoLC-MS/MS
Purification of nucleoïds from Deinococci<br />
purification<br />
trypsin<br />
Optimization<br />
of several<br />
purification steps<br />
4 differents samples<br />
(from the same protocole)<br />
2 different measurements<br />
Shotgun analysis<br />
Free-label quantification<br />
205 proteins quantified<br />
(700 proteins d<strong>et</strong>ected)
Thermococcus gammatolerans…<br />
DNA repair system<br />
General m<strong>et</strong>abolism<br />
Shotgun analysis<br />
2.0 Mbp<br />
114 nanoLC-MS/MS<br />
442 916 MS/MS spectra<br />
167 497 assignments (40%)<br />
10841 unique pepti<strong>des</strong><br />
p25)<br />
1 076 proteins (0.5% FP)<br />
(50% of the theor<strong>et</strong>ical proteome [2157])<br />
155 protein N-termini<br />
(22 ac<strong>et</strong>ylated)<br />
20 N-ter corrected
Many thanks to...<br />
Elodie Sahinovic<br />
Mathieu Baud<strong>et</strong><br />
Philippe Guérin<br />
Bernard Fernandez<br />
Alain Dedieu<br />
Jean Armengaud<br />
iBEB-SBTN-LBSP<br />
(Marcoule)<br />
But also… Charles,<br />
Véronique, Jean-Charles,<br />
… and for your<br />
kind attention !<br />
Suzanne Sommer<br />
Magali Toueille<br />
IGM<br />
(Orsay)<br />
Arjan de Groot<br />
Rémi Dulermo<br />
Laurence Blanchard<br />
Mohamed Barakat<br />
Philippe Ort<strong>et</strong><br />
Wafa Achouak<br />
Thierry Heulin<br />
iBEB-SBVME-LEMiRE<br />
(Cadarache)<br />
Yvan Zivanovic<br />
Arnaud Lagorce<br />
Fabrice Confalonieri<br />
IGM<br />
(Orsay)<br />
Proj<strong>et</strong> Blanc: <strong>Deinococcus</strong>
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