1. Introduction Advances in 2D gel techniques Mass spectrometry in proteomics Edman Degradation Identification of proteins Peptide proteome Membrane proteins Minor proteins AM PM

2. “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 biology Prote PROTEins ome Expressed by a genome Tissue Secreted into a physiological fluid

3. Complexity of the protein world DNA : 4 Nucleotides Proteins : 20 amino acids Post-translational modifications PhosphorylationMaturationGlycosylation… 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

4. “High-throughput” mapping Separation of all proteins 2-DE electrophoresis Protein-interaction mappingDifferential comparison Control CellsApoptotic 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

5. Proteome and technology Proteomic research required to separated large numbers of proteins, To identify them, and to study their modifications. Human genome 30000 genes E.Coli 4285 genes Saccharomyces cerevisiae 5885 genes Prefractionation Sample Prep Protein Separation Identification Study Of modifications Bioinformatics 2-D Electrophoresis Automatisation

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

7. Increase of the resolution by using of gradient pH of 1 unit 5000 spots 2500 spots

8. 2-DE gels analysis 2-DE software PDQUEST, MELANIE,Z3……

11. Identification and characterization of proteins Immunodetection Using antibodies to detect one specific protein Microsequence analysis by Edman degradation (Chemistry reaction) Sensibility : >250fmoles More expensive 15h for about 15 amino acids Determination of about 80 amino acids (unknown genome) Identification by mass spectrometry : a revolution Sensibility : <1fmole By automatisation : 100 proteins per day if known genome

12. Microsequence analysis by Edman degradation Electroblotting on a inert membrane (PVDF polyvinylidene difluoride) The spot is cut Automatic Edman degradation

13. Edman Reaction

14. PTH amino acids standard

15. Proteins blocked at their N-terminal end >50 % eucaryotes 10-20% procaryotes

16. 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 gi|3914462|sp|O42897|PSD3_SCHPO16 gi|9631161|ref|NP_047943.1|16 gi|2496437|sp|P75153|Y43C_MYCPN16 gi|7441762|pir||G0174716 gi|4585307|gb|AAD25372.1|AF119821_115 gi|6912258|ref|NP_036202.1|15 gi|7594623|emb|CAB88109.1|15 gi|12275312|dbj|BAB21018.1|15 gi|8118083|gb|AAF72882.1|AAF7288215 gi|7708392|emb|CAB90108.1|15 gi|4093196|gb|AAD03057.1|15 gi|6753146|ref|NP_033860.1|15 gi|12742385|ref|XP_009552.2|15 gi|12275308|dbj|BAB21017.1|15 gi|8118082|gb|AAF72881.1|AAF7288115 gi|7573540|emb|CAB87588.1|15 gi|12061185|gb|AAG45474.1|15 gi|2842424|emb|CAA11679.1|15 gi|12730688|ref|XP_003499.2|15 gi|28590|emb|CAA23753.1|15 gi|6013427|gb|AAF01333.1|AF190168_115 gi|28592|emb|CAA23754.1|15 gi|2500992|sp|P77892|SYGA_MORCA15 gi|4502027|ref|NP_000468.1|15 gi|7460098|pir||T0507615 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|7441762|pir||G0174725 gi|6013427|gb|AAF01333.1|AF190168_125 gi|28590|emb|CAA23753.1|25 gi|4502027|ref|NP_000468.1|25 gi|28592|emb|CAA23754.1|25 gi|12730688|ref|XP_003499.2|25 gi|178345|gb|AAA98798.1|25 gi|11513969|pdb|1E7E|A25 gi|229552|prf||754920A24 gi|418694|pir||ABBOSgi|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 58424 gi|113580|sp|P02770|ALBU_RAT24 gi|2190337|emb|CAA41735.1|24 gi|1351907|sp|P02769|ALBU_BOVIN24 gi|113582|sp|P14639|ALBU_SHEEP23 gi|2492797|sp|Q28522|ALBU_MACMU23 gi|543794|sp|P35747|ALBU_HORSE23 11 aa DAHKSEVAHR

17. 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 = 5.7 Mw = 66000 delta-pI = 0.25 delta-Mw = 13200 OS or OC = ALL KW keyword = ALL Display the N-terminal sequence. Tag = DAHKS SWISS-PROTTrEMBL Number of proteins found in the specified pI/Mw ranges 21644788 matching the tag 'DAHKS'10 not matching the tag 'DAHKS'21634788 Scan in SWISS-PROT database (92703 entries) 2164 proteins found in the specified pI/Mw ranges --- Results with tagging: 1 found The number before the sequence indicates the position in the mature protein where your tag DAHKS has been found (first occurrence). If the protein displayed results from the processing of a precursor, the position of the tag in the precursor polypeptide will be given in brackets. The sequence tag itself is printed in lowercase. --- --- Results without tagging: 2163 found (Printing the N-terminal sequence) --- SERUM ALBUMIN PRECURSOR pI: 5.67, MW: 66472.21 SERUM ALBUMIN. dahksEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHV....