Protein sequencing

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Various methods and strategies applied in protein sequencing

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Protein sequencing

  1. 1. By- Vikas Kr. Singh, M. Sc. Biotechnology vikasbiotech10@gmail.com vikasbiotech10@gmail.com
  2. 2. 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 vikasbiotech10@gmail.com
  3. 3. 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. vikasbiotech10@gmail.com
  4. 4. 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 vikasbiotech10@gmail.com
  5. 5. 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.vikasbiotech10@gmail.com
  6. 6. 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) vikasbiotech10@gmail.com
  7. 7. 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. vikasbiotech10@gmail.com
  8. 8. • 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) vikasbiotech10@gmail.com
  9. 9. • 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) vikasbiotech10@gmail.com
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  14. 14. How to overcome with the by-products formed in Edman degradation without losing the polypeptide chain? vikasbiotech10@gmail.com
  15. 15. Solid-phase matrix-the Merrifield resin vikasbiotech10@gmail.com
  16. 16. 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. vikasbiotech10@gmail.com
  17. 17. 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. vikasbiotech10@gmail.com
  18. 18. Protection of sulfyhydryl groups vikasbiotech10@gmail.com
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  20. 20. Separation and Molecular Weight Determination 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.vikasbiotech10@gmail.com
  21. 21. 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 vikasbiotech10@gmail.com
  22. 22. • 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 vikasbiotech10@gmail.com
  23. 23. 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. vikasbiotech10@gmail.com
  24. 24. Enzymatic Fragmentation vikasbiotech10@gmail.com
  25. 25. Chemical Fragmentation Methods • Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end forming a homoserine lactone vikasbiotech10@gmail.com
  26. 26. 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. vikasbiotech10@gmail.com
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  28. 28. 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. vikasbiotech10@gmail.com

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