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Introduction

Introduction

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Introduction

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

  2. Prote ome PROTEins Expressed by a genome Proteome and Proteomic Research “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 Tissue Secreted into a physiological fluid

  3. Environmental stress Complexity of the protein world DNA : 4 Nucleotides Proteins : 20 amino acids The famous dogmas of biology, one gene –one protein hypothesis is no tenable RNA Splicing Post-translational modifications …… Phosphorylation Maturation Glycosylation… Proteome, unlike the genome, is not a fixed feature of an organism

  4. Separation of all proteins 2-DE electrophoresis Multiprotein complexes purified by affinity (antibodies) Complexes separated by electrophoresis 1-D or 2-DE or 2-D nano HPLC Control Cells Apoptotic Cells Identification of all proteins Identification of proteins Databases 2-DE databases Accessible on the Net Bioinformatic Identification of proteins Proteomic Research “High-throughput”mapping Differential comparison Protein-interaction mapping

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

  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……

  9. Immunodetection Using antibodies to detect one specific protein Identification and characterization of proteins 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

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

  11. Edman Reaction

  12. PTH amino acids standard

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

  14. 6 aa >100 proteins DAHKSE 11 aa DAHKSEVAHR 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 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 Identification by comparison with databases N-terminal sequence is compared to the ones included in protein (SwissProt) or gene databases (Genbank) Blast, Tblast, Fasta and TFASTa Amino acids number required for identification : 10-15 aa

  15. SWISS-PROT TrEMBL Number of proteins found in the specified pI/Mw ranges 2164 4788 matching the tag 'DAHKS' 1 0 not matching the tag 'DAHKS' 2163 4788 TagIdent If you have pI and MW, 4-5 amino acids are sufficient Results from TagIdent 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.--- 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 ---Results without tagging: 2163 found(Printing the N-terminal sequence)--- SERUM ALBUMIN PRECURSOR pI: 5.67, MW: 66472.21 SERUM ALBUMIN. dahksEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHV... .