Step 1: Separation of Polypeptide Chains: If the protein of interest is a heteromultimer (composed of more than one type of polypeptide chain), then the protein must be dissociated and its component polypeptide subunits must be separated from one another and sequenced individually. Subunit associations in multimeric proteins are typically maintained solely by noncovalent forces, and th erefore most multimeric proteins can usually be dissociated by exposure to pH extremes, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All of these treatments disrupt polar interactions such as hydrogen bonds both within the protein molecule and between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or charge. Occasionally, heteromultimers are linked together by interchain S--S bridges. In such instances, these cross-links must be cleaved prior to dissociation and isolation of the individual chains. The methods described under step 2 are applicable for this purpose. Step 2: Cleavage of Disulfide Bridges A number of methods exist for cleaving disulfides. An important consideration is to carry out these cleavages so that the original or even new S--S links do not form. Oxidation of a disulfide by performic acid results in t he formation of two equivalents of cysteic acid. Because these cysteic acid side chains are ionized SO3 groups, electrostatic repulsion (as well as altered chemistry) prevents S--S recombination. Alternatively, sulfhydryl compounds such as 2-mercaptoethanol readily reduce S--S bridges to re generate two cysteine-SH side chains. However, these SH groups recombine to re-form either the original disulfide link or, if otherfree Cys-SHs are available, new disulfide links. To prevent this, SOS reduction must be followed by treatment with alkylating agents such as iodoacetate or 3-bromopropylamine, which modify the SH groups and block disulfide bridge formation.
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If the protein of interest is a heteromultimer (composed of more than one type of polypeptide
chain), then the protein must be dissociated and its component polypeptide subunits must be
separated from one another and sequenced individually. Subunit associations in multimeric proteins are typically maintained solely by noncovalent forces, and therefore most multimeric
proteins can usually be dissociated by exposure to pH extremes, 8 M urea, 6 M guanidinium
hydrochloride, or high salt concentrations. (All of these treatments disrupt polar interactions
such as hydrogen bonds both within the protein molecule and between the protein and the
aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one
another on the basis of differences in size and/or charge. Occasionally, heteromultimers are
linked together by interchain S--S bridges. In such instances, these cross-links must be
cleaved prior to dissociation and isolation of the individual chains. The methods described
under step 2 are applicable for this purpose.
Step 2: Cleavage of Disulfide Bridges
A number of methods exist for cleaving disulfides. An important consideration is to carry out
these cleavages so that the original or even new S--S links do not form. Oxidation of a
disulfide by performic acid results in the formation of two equivalents of cysteic acid.
Because these cysteic acid side chains are ionized SO3 groups, electrostatic repulsion (as well
as altered chemistry) prevents S--S recombination. Alternatively, sulfhydryl compounds such
as 2-mercaptoethanol readily reduce S--S bridges to regenerate two cysteine-SH side chains.
However, these SH groups recombine to re-form either the original disulfide link or, if other
free Cys-SHs are available, new disulfide links. To prevent this, SOS reduction must be
followed by treatment with alkylating agents such as iodoacetate or 3-bromopropylamine,
which modify the SH groups and block disulfide bridge formation.
Amino terminal end of polypeptide chain determined by either of three methods
namely sangar method, Edmann’s method and dansyl chloride method. In sanger method,
Fluorodinitrobenzene used. Polypeptide reacts with FDNB to form Dinitrophenol complex
with polypeptide. Subsequent analysis releases a colored dinitrophenol labeled aminoterminal amino acid, which can be identified by its characteristic migration rate on thin-layer
chromatography or paper electrophoresis. In Edman degradation method, Phenyl isothio
cyanate (PIT) is used as reagent. First the polypeptide is rected with phenyl isothio cyanate
to forma polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino
terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected
spectrophotometrically. Upto this stage, this method is used to determine N-terminal
aminoacid. This method can also be extended to determine polypeptide sequence. For this,
after the release of PTH, the remaining intact polypeptide, shortened by one amino acid, is
then ready for further cycles of this procedure. Dansyl chloride is also used to determine N-
terminal amino acid. This method is more sensitive because of fluorescence measurement.
There are two methods used for C-terminal determination. They are Hydrazine
method and carboxy peptidase method. Chemical methods for carboxy end-group
determination are considerably less satisfactory. Treatment of the peptide with anhydrous
hydrazine at 100*C results in conversion of all the amino acid resiudes to amino acid
hydrazides except for the carboxy-terminal residue, which remains as the free amino acid and
can be isolated and identified chromatographically. Alternatively, polypeptide can be
subjected to limited breakdown with the enzyme carboxypeptidase. Carboxypeptidases areenzymes that cleave amino acid residues from the C-termini of polypeptides in a successive
fashion. Four carboxypeptidases are in general use: A, B, C, and Y. Carboxypeptidase A
(from bovine pancreas) works well in hydrolyzing the Cterminal peptide bond of all residues
except proline, arginine, and lysine. The analogous enzyme from hog pancreas,
carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a
mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline.
Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-
terminal residue. Because the nature of the amino acid residue at the end often determines
the rate at which it is cleaved and because these enzymes remove residues successively, care
must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an
automated protocol analogous to that used in Edman sequenators.
The aim at this step is to produce fragments useful for sequence analysis. The cleavage
methods employed are usually enzymatic, but proteins can also be fragmented by specific or
nonspecific chemical means (such as partial acid hydrolysis). Fragmentation can be
achieved by the use of endopeptidases, which are enzymes that catalyze polypeptide chain
cleavage at specific sites in the protein. The specificity of four endopeptidases commonly for
this purpose are Pepsin, Trypsin, Chymotrypsin and Papain. Another specific chemicalmethod for polypeptide chain cleavage involves reaction with cyanogens bromide. This
reaction cleaves specifically at the methionine residues, with the accompanying conversion of
free carboxyl-terminal methionine to homoserine lactone. For sequencing protein minimum
two proteolytic factors should be selected and two sets of fragements should be prepared.
Peptides resulting from cleavage of the intact protein are generally separated by
column chromatography. The isolated peptides may then be analyzed to determine their
sequence. There two methods recently used for this purpose, they are Edman degradation
and tandem mass spectrometer. Devices called sequenators are available that automate
Edman degradation procedure. The success of these devices depends in large part on the
technical innovation of covalently linking the peptide to be sequenced to glass
beads. Attachment of peptide through its carboxy-terminal group to this immobile phase
facilitates the complete removal of potentially conataminating reaction procedure during
successive stages of the degradation. Thus with the help of this sequenators, peptide
fragements are sequenced.
The other method used for protein sequencing is tandem mass spectrometer. Tandem
MS (or MS/MS) allows sequencing of proteins by hooking two mass spectrometers in tandem.
The first mass spectrometer is used to separate oligopeptides from a protein digest and then to
select in turn each of these oligopeptides for further analysis. A selected ionized oligopeptideis directed toward the second mass spectrometer; on the way, this oligopeptide is fragmented
by collision with helium or argon gas molecules, and the collection of fragments is analyzed
by the second mass spectrometer. Fragmentation occurs primarily in the peptide bonds
linking successive amino acids in the oligopeptide, so the fragments created represent a
nested set of peptides that differ in size by one amino acid residue. The fragments differ in
mass by 56 atomic mass units (the mass of the peptide backbone atoms (NH-CH-CO)) plus
the mass of the R group at each position, which ranges from 1 atomic mass unit (Gly) to 130
(Trp). MS sequencing has the advantages of very high sensitivity, fast sample processing, and
the ability to work with mixtures of proteins. Subpicomoles (less than 10_12 moles) of
peptide can be analyzed. However, in practice, tandem MS is limited to rather short
sequences (no longer than 15 or so amino acid residues).