BY:KANIKA SABHARWAL

MTECH BIOINFORMATICS

DEFINITIONProtein sequencing is determining the

amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any non-peptide molecules.

HISTORY

The first protein sequencing was achieved by Frederic Sanger in 1953.

He determined the amino acid sequence of bovine insulin

He took 10 years and approximately 100 grams of proteins

Sanger was awarded the Nobel Prize in 1958

METHODS FOR PROTEIN SEQUENCING

EDMAN DEGRADATIONMASS SPECTROSCOPYKNOWN DNA SEQUENCE

END GROUP ANALYSISProtein has C as well N terminalIdentifying these groups can establish the

number of chemically distinctive polypeptides in a protein

Identification of N-terminus of protein

1. Sanger’s reagent 1-fluoro-2,4-

dinitrobenzene (FDNB)2. dansyl chloride and

dabsyl chloride

IDENTIFICATION OF C-TERMINAL AMINO ACID

CARBOXYPEPTIDASE- hydrolytic excision of

polypeptide C-terminal residue

CARBOXYPEPTIDASE A- intestinal digestive

enzyme, does not cleave C-terminal arginine or lysine; or residues next to proline

CARBOXYPEPTIDASE B- hydrolyses only C-

terminal arginine or lysine residues, but only if they are not proceeded by proline

CARBOXYPEPTIDASES CLEAVE THE C-TERMINAL RESIDUES

BREAKING DISULFIDE BONDSDisulfide bonds interfere with the sequencing procedure.

DISADVANTAGE OF PERFORMIC ACID IN CLEAVING DISULPHIDE BONDS

Performic acid oxidizes methionine residues and partially destroys the indole side chain of tryptophan

ADVANTAGE OF USING INDOLE ACETIC ACID

Prevents the reformation of disulphide bonds by alkylating the sulphydryl group

• The overall accuracy of amino acid sequencing generally declines as the length of the polypeptide increases protein is cleaved into a set of specific fragments by chemical or enzymatic methods

• If any disulfide bonds are present, they must be broken.

• Each fragment is purified, then sequenced by the Edman procedure

• Finally, the order in which the fragments appear in the original protein is determined and disulfide bonds (if any) are located

PROTEIN CLEAVAGE

EDMAN DEGRADATIONCyclic degradation of peptides based on

the reaction of Phenylisothiocyanate with the free amino group of the N-terminal residue such that amino acids are removed one at a time and identified as their phenylthiohydantoin derivatives

1. Phenylisothiocyanate reacts with the N-terminal amino acid of peptides

2. Adduct undergoes cyclization with cleavage of the peptide linkage under acidic conditions

3. After rearrangement the resulting phenylthioanhydantion of the N-terminal amino acid can be identified

4. Procedure repeated on the shortened peptide chain to identify the amino acid residue in the second position

SEPARATION OF PTH-AMINO ACIDS

PTH-amino acids can be rapidly separated by high-pressure liquid chromatography (HPLC). In this HPLC profile, a mixture of PTH-amino acids is clearly resolved into its components.

An unknown amino acid can be identified by its elution position relative to the known ones.

ORDERING PEPTIDE FRAGMENTSThe amino acid sequences of each fragment

obtained by the two cleavage procedures are examined

Overlapping peptides obtained from the second fragmentation yield the correct order of the peptide fragments produced in the first

Edman’s degradation has been a tremendous asset in protein sequencing, however, for larger proteins recombinant DNA technology is now being used.

SEQUENCING USING DNA

PROTEIN SEQUENCING BY MS

PROTEIN DATABASES PIR (Protein Information Resource)MIPS (Marstinsried Institute For Protein

Sequences)SWISS-PROT TrEMBL (Translated European Molecular

Biology Lab)

APPLICATIONS OF PROTEIN SEQUENCING

Structural and functional analyses of proteinDiscovery of new protein marker for

diagnostic purposesTo develop a novel molecular drug target for

drug discoveryStudying proteins on large scale for

automation and new technologies

Thanku………….