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)