protein sequencing
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PROTEIN SEQUENCING
By-Vikas Kr. Singh, M. Sc. Biotechnologyvikasbiotech10@gmail.com
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First Sequence
• The first protein sequencing was achieved by Frederic Sanger in 1953. He determined the amino acid sequence of bovine insulin.
• Sanger was awarded the Nobel Prize in 1958
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Strategy for protein sequencing• Determine number of polypeptide chains (subunits).• Determine number of disulfide bonds (inter- and
intra-chain).• Determine the amino acid composition of each
polypeptide chain.• If subunits are too large, fragment them into shorter
polypeptide chains.• Sequence each fragment using the Edman
degradation method.• Complete the sequence by comparing overlaps of
different sets of fragments.
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End-group Analysis• Number of chains can be determine by
identifying the number of N- and C-terminal.
• N-terminal analysis– Dansyl chloride or FDNB method– Phenylisothiocynate (PITC)/ Edman reagent– Aminopeptidase
• C-terminal analysis– carboxypeptidase
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N-terminal Analysis with Dansyl Chloride• Reagent: 1-dimethyl
aminophthalene-5-sulfonyl chloride (dansyl chloride)
• Dansyl polypeptide chain is prepared
• Acidic hydrolysis liberates all amino acid and the N terminal dansyl amino acid
• Amino acids are separated• Fluorescence of the dansyl
amino acid is detected• Type of aa is obtained from
comparison with standard dansylated amino acids.
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N-terminal Analysis Edman (Degradation)
• Nucleophilic attack on phenyl isothiocyanate (PITC), the Edman reagent, under mild alkaline conditions (Nmethylpiperidine/ water/ methanol).
• Formation of a phenylthiocarbamyl derivative (PTC-peptide)
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N-terminal Analysis Edman (Degradation)• Anhydrous trifluoro acetic
acid (TFA) used to cleave the terminal amino acid in the form of a thiozolinone derivative leaving the other peptide bonds intact.
• The thiozolinone (TZ) derivative is extracted in an organic solvent (e.g. N-butyl chloride)
• Peptide cleaved carries a free amino terminus.
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• The TZ is extracted into an organic solvent and treated with an acid (25 % TFA/water) to form phenylthiohydantoin (PTH) derivative.
• PTH is detected from UV absorption at 296 nm
N-terminal Analysis Edman (Degradation)
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• PTH amino acid is separated from the other components by chromatography or electrophoresis.
• The terminal amino is identified according to retention time or mass.
• This sequence can be repeated to identify all amino acid in short peptide chains (40-60 amino acid long).
N-terminal Analysis Edman (Degradation)
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How to overcome with the by-products formed in Edman
degradation without losing the polypeptide chain?
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Solid-phase matrix-the Merrifield resin
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N- and C- terminal Analysis-Exopeptidase Method
• Exopeptidases cleave the terminal residue of a polypeptide chain.
• Aminopeptidases cleave the N-terminal residues.
• Carboxypeptidases cleave the C-terminal residues.
• Further analyzed by amino acid analyzer.
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Disulfide Bond Cleavage
• Disulfides are reduced to thiol with dithiothreitol (DTT) or 2- mercaptoethanol.
• Thiols are treated with alkylating agents (e.g. iodoacetic acid) to prevent the re-oxidation during subsequent steps.
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Protection of sulfyhydryl groups
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Separation and Molecular WeightDetermination of Subunits
• Traditional Methods:-– SDS-PAGE, SEC, or RP-HPLC used to separate the subunits after cleavage of disulfide bonds.– Mw standards and a calibration curve are used
to determine the Mw.– The approximate no. of amino acids estimated from the Mw of the subunit using 110 Da as the average molar mass for each amino acid.
• Recent methods– MALDI:- more accurate and fast.
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Amino Acid composition• Strategy:-
-Hydrolysis followed by separation and identification• Acid catalyzed hydrolysis
-6M HCl/ 100-120°C/ 24 h (in oxygen free environment to prevent oxidation of SH groups)-Some residues are degraded under these harsh conditions
• Base catalyzed hydrolysis-4 M NaOH /100°C/ 4-8 hours-Arg, Cys, Ser and Thr are decomposed and other amino acids are deaminated and racemized.-Used mainly to determine Trp which is extensively degraded under acid catalyzed hydrolysis
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• Enzymatic hydrolysis-By exo- and endopeptidases-A combination of endo and exopeptidases must be used to hydrolyze all the peptide bonds.
• Separation-Individual amino acids in hydrolyzed mixture can be separated by RP-HPLC or CE and identified according to retention time
Amino Acid composition
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Cleavage of Specific Peptide Bonds• Direct sequencing is applicable to peptides that
have up to about 50 residues only.• Problems occur after lengthy reactions
-Incomplete reactions-Accumulation of impurities from side reactions
• Solution:- Use enzymes to fragment the polypeptide chain.-Proteolytic enzymes: - endopeptidases and
- exopeptidases.
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Enzymatic Fragmentation
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Chemical Fragmentation Methods• Cyanogen bromide (CNBr) specifically cleaves
Met residues at the C-end forming a homoserine lactone
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Ordering of Peptide Fragments• Compare amino acid sequence of one set of
peptide fragments with the sequence of a second set of fragments obtained using different cleavage points.
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Determination of Disulfide Bond Position
• Digest polypeptide chain(s)• Run 2D gel of mixture of fragments using same
conditions in both dimension• After separation in the first dimension, the matrix
is exposed to performic acid which cleaves all possible disulfide bonds
• Separation in the second dimension is performed.
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