Proteomic Research
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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. Prote. ome. PROTE ins. Expressed by a gen ome.

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Introduction

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


Introduction

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


Introduction

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


Introduction

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


Introduction

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


Introduction

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


Introduction

Increase of the resolution by using of gradient pH of 1 unit

5000 spots

2500 spots


Introduction

2-DE gels analysis

2-DE software PDQUEST, MELANIE,Z3……


Introduction

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


Introduction

Electroblotting on a inert membrane (PVDF polyvinylidene difluoride)

The spot is cut

Automatic Edman degradation

Microsequence analysis by Edman degradation


Introduction

Edman Reaction


Introduction

PTH amino acids standard


Introduction

Proteins blocked at their N-terminal end

>50 % eucaryotes

10-20% procaryotes


Introduction

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


Introduction

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...

.


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