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Transcript 
Introduction
AM
Advances in 2D gel techniques
Identification of proteins
Mass spectrometry in proteomics
Edman Degradation
Peptide proteome
Membrane proteins
Minor proteins
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 ome
PROTEins Expressed by a genome
Tissue
Secreted into a physiological fluid
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
Environmental stress
“High-throughput” mapping
Differential comparison
Separation of all proteins
2-DE electrophoresis
Multiprotein complexes purified by
affinity (antibodies)
Control Cells
Identification of all proteins
Databases
2-DE databases
Accessible on the Net
Protein-interaction mapping
Apoptotic Cells
Complexes separated by
electrophoresis 1-D or 2-DE or 2D nano HPLC
Identification of proteins
Bioinformatic
Identification of proteins
Proteome and technology
Prefractionation
Sample Prep
Automatisation
Protein Separation
2-D Electrophoresis
Proteomic research required
to separated large numbers
of proteins,
To identify them, and to
study their modifications.
Human genome
30000 genes
Identification
Study
Of
modifications
Bioinformatics
Saccharomyces cerevisiae
5885 genes
E.Coli
4285 genes
Two-dimensional electrophoresis: The state of the art and future directions
5000 spots
2500 spots
Increase of the resolution by using of gradient pH of 1 unit
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)
Blast, Tblast, Fasta and TFASTa
Amino acids number required for identification : 10-15 aa
6 aa >100 proteins DAHKSE
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
11 aa
DAHKSEVAHR
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
TagIdent
If you have pI and MW, 4-5 amino acids are sufficient
Results from TagIdent
Scan in SWISS-PROT database (92703 entries)
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
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.
--SWISS-PROT
Number of proteins found in the specified pI/Mw ranges
4788
0
4788
matching the tag 'DAHKS'
not matching the tag 'DAHKS'
--Results without tagging: 2163
found
(Printing the N-terminal sequence)
SERUM ALBUMIN PRECURSOR
pI: 5.67, MW: 66472.21 SERUM ALBUMIN.
dahksEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHV...
.
2164
1
2163
TrEMBL
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