Introduction

Advances in 2D gel techniques

Mass spectrometry in proteomicsEdman Degradation

Identification of proteins

Peptide proteome

Membrane proteins

Minor proteins

AM

PM

“The sequencing of the human genome is a monumental achievement that will radically reshape biology and medicine. Now we begin the challenge of adding interpretation and meaning to the DNA code. “

Post-Genomic area

Proteomic holds a key position in the new biologyProte

PROTEins

ome

Expressed by a genome

TissueSecreted into a physiological fluid

Complexity of the protein world

DNA : 4 Nucleotides

Proteins : 20 amino acids

Post-translational modifications

Phosphorylation Maturation Glycosylation…

Proteome, unlike the genome, is not a fixed feature of an organism

Environmental stress

The famous dogmas of biology, one gene –one protein hypothesis is no tenable

……

RNA Splicing

“High-throughput” mapping

Separation of all proteins2-DE electrophoresis

Protein-interaction mappingDifferential comparison

Control Cells Apoptotic Cells

Identification of proteins

Multiprotein complexes purified by affinity (antibodies)

Complexes separated by electrophoresis 1-D or 2-DE or 2-

D nano HPLC

Identification of proteins

Databases 2-DE databases

Accessible on the Net

Identification of all proteins

Bioinformatic

Proteome and technology

Proteomic research required to separated large numbers of proteins,To identify them, and to study their modifications.

Human genome30000 genes

E.Coli4285 genes

Saccharomyces cerevisiae5885 genes

PrefractionationSample Prep

Protein Separation

IdentificationStudy

Ofmodifications

Bioinformatics

2-D Electrophoresis

Autom

atisation

Two-dimensional electrophoresis: The state of the art and future directions

Increase of the resolution by using of gradient pH of 1 unit

5000 spots

2500 spots

2-DE gels analysis2-DE software PDQUEST, MELANIE,Z3……

Identification and characterization of proteins

ImmunodetectionUsing antibodies to detect one specific protein

Microsequence analysis by Edman degradation (Chemistry reaction)

Sensibility : >250fmolesMore expensive

15h for about 15 amino acidsDetermination of about 80 amino acids (unknown genome)

Identification by mass spectrometry : a revolution

Sensibility : <1fmoleBy automatisation : 100 proteins per day if known genome

Microsequence analysis by Edman degradation

Electroblotting on a inert membrane (PVDF

polyvinylidene difluoride)

The spot is cut

Automatic Edman degradation

Edman Reaction

PTH amino acids standard

Proteins blocked at their N-terminal end

>50 % eucaryotes10-20% procaryotes

Identification by comparison with databases

N-terminal sequence is compared to the ones included in protein (SwissProt) or gene databases (Genbank)

Amino acids number required for identification : 10-15 aa

Blast, Tblast, Fasta and TFASTa

Score E Sequences producing significant alignments: (bits) Value gi|3914462|sp|O42897|PSD3_SCHPO PROBABLE 26S PROTEASOME REG... 16 67872 gi|9631161|ref|NP_047943.1| gp52 [Bacteriophage phi-C31] >g... 16 67872 gi|2496437|sp|P75153|Y43C_MYCPN HYPOTHETICAL LIPOPROTEIN MG... 16 67872 gi|7441762|pir||G01747 albumin homolog - human (fragment) >... 16 115771 gi|4585307|gb|AAD25372.1|AF119821_1 (AF119821) attractin [M... 15 129035 gi|6912258|ref|NP_036202.1| attractin; attractin (with dipe... 15 129035 gi|7594623|emb|CAB88109.1| (AL132773) dJ741H3.1.1 (attracti... 15 129035 gi|12275312|dbj|BAB21018.1| (AB038388) attractin [Rattus no... 15 129035 gi|8118083|gb|AAF72882.1|AAF72882 (AF218915) secreted attra... 15 129035 gi|7708392|emb|CAB90108.1| (AL132773) dJ741H3.1.2 (KIAA0548... 15 129035 gi|4093196|gb|AAD03057.1| (AF106861) attractin-2 [Homo sapi... 15 129035 gi|6753146|ref|NP_033860.1| attractin [Mus musculus] >gi|44... 15 129035 gi|12742385|ref|XP_009552.2| attractin [Homo sapiens] 15 129035 gi|12275308|dbj|BAB21017.1| (AB038387) attractin [Rattus no... 15 129035 gi|8118082|gb|AAF72881.1|AAF72881 (AF218915) membrane attra... 15 129035 gi|7573540|emb|CAB87588.1| (AL121965) dJ161I14.1 (RNA helic... 15 132306 gi|12061185|gb|AAG45474.1| (AY013288) ASC-1 complex subunit... 15 132306 gi|2842424|emb|CAA11679.1| (AJ223948) RNA helicase [Homo sa... 15 132306 gi|12730688|ref|XP_003499.2| albumin precursor [Homo sapiens] 15 139098 gi|28590|emb|CAA23753.1| (V00494) reading frame HSA [Homo s... 15 140264 gi|6013427|gb|AAF01333.1|AF190168_1 (AF190168) serum albumi... 15 140264 gi|28592|emb|CAA23754.1| (V00495) serum albumin [Homo sapiens] 15 141439 gi|2500992|sp|P77892|SYGA_MORCA GLYCYL-TRNA SYNTHETASE ALPH... 15 141439 gi|4502027|ref|NP_000468.1| albumin precursor; PRO0883 prot... 15 142624 gi|7460098|pir||T05076 hypothetical protein T6K21.80 - Arab... 15 143819

6 aa >100 proteins DAHKSE

Score E Sequences producing significant alignments: (bits) Value gi|7441762|pir||G01747 albumin homolog - human (fragment) >... 25 154 gi|6013427|gb|AAF01333.1|AF190168_1 (AF190168) serum albumi... 25 158 gi|28590|emb|CAA23753.1| (V00494) reading frame HSA [Homo s... 25 160 gi|4502027|ref|NP_000468.1| albumin precursor; PRO0883 prot... 25 161 gi|28592|emb|CAA23754.1| (V00495) serum albumin [Homo sapiens] 25 161 gi|12730688|ref|XP_003499.2| albumin precursor [Homo sapiens] 25 167 gi|178345|gb|AAA98798.1| (M12523) alloalbumin Venezia [Homo... 25 173 gi|11513969|pdb|1E7E|A Chain A, Human Serum Albumin Complex... 25 186 gi|229552|prf||754920A albumin [Bos taurus] 24 260 gi|418694|pir||ABBOS serum albumin precursor [validated] - ... 24 348 gi|113580|sp|P02770|ALBU_RAT SERUM ALBUMIN PRECURSOR >gi|72... 24 351 gi|2190337|emb|CAA41735.1| (X58989) serum albumin [Bos taur... 24 375 gi|1351907|sp|P02769|ALBU_BOVIN SERUM ALBUMIN PRECURSOR >gi... 24 385 gi|113582|sp|P14639|ALBU_SHEEP SERUM ALBUMIN PRECURSOR >gi|... 23 436 gi|2492797|sp|Q28522|ALBU_MACMU SERUM ALBUMIN PRECURSOR >gi... 23 482 gi|543794|sp|P35747|ALBU_HORSE SERUM ALBUMIN PRECURSOR >gi|... 23 584

11 aa DAHKSEVAHR

If you have pI and MW, 4-5 amino acids are sufficient

TagIdent

Results from TagIdent

The search in SWISS-PROT and TrEMBL has been launched with the following values:pI = 5.7Mw = 66000delta-pI = 0.25delta-Mw = 13200OS or OC = ALLKW keyword = ALLDisplay the N-terminal sequence.Tag = DAHKS

SWISS-PROT TrEMBL

Number of proteins found in the specified pI/Mw ranges 2164 4788matching the tag 'DAHKS' 1 0

not matching the tag 'DAHKS' 2163 4788

Scan in SWISS-PROT database (92703 entries)

2164 proteins found in the specified pI/Mw ranges---Results with tagging: 1 foundThe number before the sequence indicates the position in themature protein where your tag DAHKS has been found (first occurrence).If the protein displayed results from the processing of aprecursor, the position of the tag in the precursor polypeptidewill be given in brackets.The sequence tag itself is printed in lowercase.---

---Results without tagging: 2163 found(Printing the N-terminal sequence)---

SERUM ALBUMIN PRECURSORpI: 5.67, MW: 66472.21 SERUM ALBUMIN.

dahksEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHV...

.