Introduction Advances in 2D gel techniques Mass spectrometry in proteomics Edman 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 biology Prote PROTEins ome Expressed by a genome Tissue Secreted into a physiological fluid

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

“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

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

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 analysis 2-DE software PDQUEST, MELANIE,Z3……

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

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 % eucaryotes 10-20% 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 : aa Blast, Tblast, Fasta and TFASTa Score E Sequences producing significant alignments: (bits) Value gi| |sp|O42897|PSD3_SCHPO PROBABLE 26S PROTEASOME REG gi| |ref|NP_ | gp52 [Bacteriophage phi-C31] >g gi| |sp|P75153|Y43C_MYCPN HYPOTHETICAL LIPOPROTEIN MG gi| |pir||G01747 albumin homolog - human (fragment) > gi| |gb|AAD |AF119821_1 (AF119821) attractin [M gi| |ref|NP_ | attractin; attractin (with dipe gi| |emb|CAB | (AL132773) dJ741H3.1.1 (attracti gi| |dbj|BAB | (AB038388) attractin [Rattus no gi| |gb|AAF |AAF72882 (AF218915) secreted attra gi| |emb|CAB | (AL132773) dJ741H3.1.2 (KIAA gi| |gb|AAD | (AF106861) attractin-2 [Homo sapi gi| |ref|NP_ | attractin [Mus musculus] >gi| gi| |ref|XP_ | attractin [Homo sapiens] gi| |dbj|BAB | (AB038387) attractin [Rattus no gi| |gb|AAF |AAF72881 (AF218915) membrane attra gi| |emb|CAB | (AL121965) dJ161I14.1 (RNA helic gi| |gb|AAG | (AY013288) ASC-1 complex subunit gi| |emb|CAA | (AJ223948) RNA helicase [Homo sa gi| |ref|XP_ | albumin precursor [Homo sapiens] gi|28590|emb|CAA | (V00494) reading frame HSA [Homo s gi| |gb|AAF |AF190168_1 (AF190168) serum albumi gi|28592|emb|CAA | (V00495) serum albumin [Homo sapiens] gi| |sp|P77892|SYGA_MORCA GLYCYL-TRNA SYNTHETASE ALPH gi| |ref|NP_ | albumin precursor; PRO0883 prot gi| |pir||T05076 hypothetical protein T6K Arab gi| |sp|O42897|PSD3_SCHPO16 gi| |ref|NP_ |16 gi| |sp|P75153|Y43C_MYCPN16 gi| |pir||G gi| |gb|AAD |AF119821_115 gi| |ref|NP_ |15 gi| |emb|CAB |15 gi| |dbj|BAB |15 gi| |gb|AAF |AAF gi| |emb|CAB |15 gi| |gb|AAD |15 gi| |ref|NP_ |15 gi| |ref|XP_ |15 gi| |dbj|BAB |15 gi| |gb|AAF |AAF gi| |emb|CAB |15 gi| |gb|AAG |15 gi| |emb|CAA |15 gi| |ref|XP_ |15 gi|28590|emb|CAA |15 gi| |gb|AAF |AF190168_115 gi|28592|emb|CAA |15 gi| |sp|P77892|SYGA_MORCA15 gi| |ref|NP_ |15 gi| |pir||T aa >100 proteins DAHKSE Score E Sequences producing significant alignments: (bits) Value gi| |pir||G01747 albumin homolog - human (fragment) > gi| |gb|AAF |AF190168_1 (AF190168) serum albumi gi|28590|emb|CAA | (V00494) reading frame HSA [Homo s gi| |ref|NP_ | albumin precursor; PRO0883 prot gi|28592|emb|CAA | (V00495) serum albumin [Homo sapiens] gi| |ref|XP_ | albumin precursor [Homo sapiens] gi|178345|gb|AAA | (M12523) alloalbumin Venezia [Homo gi| |pdb|1E7E|A Chain A, Human Serum Albumin Complex gi|229552|prf||754920A albumin [Bos taurus] gi| |pir||G gi| |gb|AAF |AF190168_125 gi|28590|emb|CAA |25 gi| |ref|NP_ |25 gi|28592|emb|CAA |25 gi| |ref|XP_ |25 gi|178345|gb|AAA |25 gi| |pdb|1E7E|A25 gi|229552|prf||754920A24 gi|418694|pir||ABBOSgi|418694|pir||ABBOS serum albumin precursor [validated] gi|113580|sp|P02770|ALBU_RAT SERUM ALBUMIN PRECURSOR >gi| gi| |emb|CAA | (X58989) serum albumin [Bos taur gi| |sp|P02769|ALBU_BOVIN SERUM ALBUMIN PRECURSOR >gi gi|113582|sp|P14639|ALBU_SHEEP SERUM ALBUMIN PRECURSOR >gi| gi| |sp|Q28522|ALBU_MACMU SERUM ALBUMIN PRECURSOR >gi gi|543794|sp|P35747|ALBU_HORSE SERUM ALBUMIN PRECURSOR >gi| gi|113580|sp|P02770|ALBU_RAT24 gi| |emb|CAA |24 gi| |sp|P02769|ALBU_BOVIN24 gi|113582|sp|P14639|ALBU_SHEEP23 gi| |sp|Q28522|ALBU_MACMU23 gi|543794|sp|P35747|ALBU_HORSE23 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 = 5.7 Mw = delta-pI = 0.25 delta-Mw = 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 matching the tag 'DAHKS'10 not matching the tag 'DAHKS' 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: SERUM ALBUMIN. dahksEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHV....