introduction advances in 2d gel techniques mass spectrometry in proteomics edman degradation...
TRANSCRIPT
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...
.