protein sequencing
TRANSCRIPT
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………….