Common point for sequencing peptides and proteins:
Procedure for determination of main structure of proteins and peptides engages the given major steps:
i) Establishing number of polypeptide chain in protein;
ii) Cleavage of disulfide bridges available;
iii) Separation and purification of individual polypeptide;
iv) Determination of amino acid composition;
v) Fragmentation of individual polypeptide to yield small peptides which can be sequenced;
vi) Separation and purification of small peptides;
vii) Determination of amino acid sequence of each of small peptides;
viii) Establishing overall sequence of each polypeptide in protein; and
ix) Establishing position of inter and intra disulfide-bridge, if any.
Failure in any of them may spell doom to final objective of sequencing peptide or a protein. We shall now consider each of the steps.
Steps to Sequencing:
i) Establishing Number of Polypeptide Chain in Protein:
Establishing number of chemically distinct polypeptides available for sequencing can be attained by recognizing N-terminal and/or Cterminal residues. To do this, peptides/protein is made to react with dansyl chloride (reagents that react specially with main amines) to yield dansylated polypeptide. Acid hydrolysis of later then yields dansylamino acid which can easily be recognized using chromatography.
Most helpful method for determination of N-terminal amino acid is method of Edman degradation. Method is termed after its inventor, Pehr Edman. Method labels and removes only aminoterminal residue from the peptide leaving all other peptide bonds. In method, phenylisothiocyanate (PITC, Edman's reagent) is made to react with N-terminal amino groups of peptide or protein under mild alkaline conditions to create phenylthiocarbamyl (PTC) adduct. Treatment of this adduct with anhydrous trifluoroacetic acid releases N-terminal residue as thiazolinone derivative, leaving other peptide bonds intact. Thiazolinone-amino acid is then extracted with organic solvent, converted to the more stable phenylthiohydantoin (PTH) by treatment with aqueous acid. PTH-amino acid is lastly recognized using advanced chromatographic method.
ii) Determination of Amino Acid Composition:
Knowledge of amino acid composition is very significant and its determination must precede its actual sequencing. Amino acid composition of the peptide is number of each kind of amino acid residue present in peptide. This is found out by complete acid (6MHCl) hydrolysis of peptide followed by quantitative analysis of liberated amino acids. It is sufficient it to mention that base (4M NaOH) and enzymatic hydrolysis is also possible.
iii) Fragmentation of Individual Polypeptide to Yield Small Peptides that can be sequenced:
This step is essential for proteins and large peptides as polypeptides longer than 40-100 residues can't be directly sequenced.
Fragmentation of longer polypeptides can be achieved by:
i) use of endopeptidases like Trypsin that cleaves peptide bonds on arboxyl terminus of Arginine and Lysine residues.
ii) Use of cyanogen bromide that specially cleaves peptide bonds after Methionine residues.
iv) Separation and Purification of the Small Peptides:
After generating peptide fragments of needed size, these fragments should be separated and purified for subsequent amino acid sequence determination.
Deciphering the Complete Primary Structure:
Once sequencing step is finished, remaining steps will be to establish right order of fragments sequenced and precise location of disulfide bonds in native peptide or protein.
i) Establishing Overall Sequence of Each Polypeptide in Protein:
Deciphering order in which all small peptide fragments generated happens in original polypeptide(s), needs comparing amino acid sequence of peptide fragments produced by say, cleavage with trypsin with those generated by say cyanogens bromide, given their specific cleavage sites overlap. At times, a third or even fourth cleavage using different cleaving reagent is essential before good overlap is attained.
ii) Establishing Position of Inter and Intra Disulfidebridge:
Identification of existing disulfide bonds begins with cleaving fresh sample of native protein under conditions which leave disulfide bonds intact and resulting fragments separated. Fragments containing cysteine residues are known by examining amino acid composition of all fragments generated. Cysteine residues with free sufhydryl group (those not creating sulfide bonds) are recognized by reacting peptides with radioactive iodoacetic acid. Cysteine-containing fragments are then subjected to Edman degradation from where location of cysteine(s) is/are readily deduced.
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