Proteins Sequencing

Chapter 1: Introduction

  • This chapter discusses the determination of the primary structure of proteins.

  • The first step is to hydrolyze the protein sample and identify the amino acids present.

    • The sample is separated and hydrolyzed.

    • The machine identifies the amino acids, such as tyrosine.

    • The amounts of each amino acid are quantified.

  • The second step is to identify the amino acid at the N-terminal and C-terminal ends of the polypeptide chain.

    • The sample is hydrolyzed again.

    • Enzymes are used to determine the specific amino acids at the ends.

  • The third step involves cleaving fragments and combining them to determine the sequence of amino acids.

  • This is the general roadmap for determining the sequence of amino acids in a polypeptide chain.

Chapter 2: Free Amino Acids

  • This chapter discusses the process of digesting proteins to obtain free amino acids.

  • Hydrolysis using enzymes is used to separate the polypeptide chain into free amino acids.

  • An amino acid analyzer is used to quantify specific amino acids, such as tyrosine and phenylalanine.

  • Chemicals like Phenyl isothiocyanate and DNFB are used to identify the N-terminal amino acid.

  • Edman degradation is a process that helps identify the N-terminal amino acid using Phenyl isothiocyanate.

  • Carboxypeptidase enzymes are used to determine the C-terminal amino acid.

Chapter 3: Phenylalanine Plus Amino

  • Kasi cleave selective for arginine at Shakalysine

    • If polypeptide chain has arginine residue, cleave

    • C terminal cleavage for step 2, Determination of terminals, n and c

  • Fragmentation of polypeptide chain

    • Enzyme chemicals used for cleavage

    • Example: Chemotrypsin cleaves phenylalanine, tyrosine, tryptophan

Chapter 4: Possible Arrangement Amino

  • BNFB used to determine n terminal

    • Product ID and P histidine

  • CNBR (Cyanogen bromide) treatment for c terminal

    • Obtain p amino acid and pentapeptide composition

    • Possible arrangement of amino acids

Chapter 5: Pentapeptide So Pentapeptide

  • Treatment with glutamine and cyanogen bromide

    • Cleaves to produce dipeptide and tripeptide

    • Kiniclib natripsin cleaves arginine and lysine

  • Possible combinations of amino acids

    • Position of methionine

    • Position of alanine, phenylalanine, histidine, lysine

Chapter 6: Conclusion

  • Cleavage using cyanogen bromide

    • Result in pentapeptide

    • Cleavage identity of amino acid

  • Cleavage using chemotrypsin

    • Cleaves phenylalanine and alanine