Biochemistry protein sequencing

Step 1.

Separation of polypeptide chains: denaturation

Denaturation is achieved with:

• Extremes of pH

• High urea concentration

• High guanidine HCl concentration

• High Salt concentration

Step 2.

Cleavage of disulfide bridges and alkylation

• Reducing agents: DTT and beta Me

• Use IoAc to prevent reformation of disulfide bonds (takes away the H in the SH and bonds to IoAc

Step 3.

N and C terminal analysis

N terminal analysis

• Edman’s reagent, phenyl isothiocyanate (PTH) - leaves new and reactive amino terminus

• removes one residue at a time (amino acid)

C terminal analysis

• uses carboxypeptidases to identify

Carboxypeptidase A

Cleaves any amino acid except Pro (proline) Arg, (arginine), and Lys (lysine)

Carboxypeptidase B

• Cleaves only works on Arginine and Lysine - no proline

Enzymatic fragmentation

• Trypsin

• Chymotrypsin

• Staphylococcal protease

Chemical fragmentation

• cyanogen bromide

Trypsin

Cleavage on the C-side of Lys, Arg (basic)

Chymotrypsin

C-side of Phe,Tyr, Trp; less so Leu (large nonpolar)

Aromatic residues

Staphylococcal protease

• C-side of Glu, Asp (acidic) in phosphate buffer (at 7)

• Specific for Glu in acetate (pH 5) and bicarbonate buffer (pH 10)