Selective isolation of free and blocked amino-terminal peptides from enzymatic digestion of proteins.
A general method for the selective isolation of free and blocked amino-terminal peptides from proteins is described. The rationale behind the methodology is based on the reasoning that if a protein, which has all its free amino groups blocked by citraconylation, is digested with a protease, all peptides, except those derived from the amino terminus, will have a free amino group. Reaction of such a digest with 1-fluoro-2,4-dinitrobenzene (Dnp-F) followed by removal of citraconyl groups by acid treatment and removal of dinitrophenyl (Dnp) groups from histidine and tyrosine side chains by thiolysis will result in dinitrophenylation of all alpha-amino groups of peptides generated from internal cleavages, leaving only peptides derived from the amino terminus without a Dnp group. The strong adsorption of Dnp groups to polystyrene is used to selectively elute the underivatized amino-terminal peptides from such a column. It is also demonstrated how selective isolation of amino-terminal peptides can be used to determine whether a protein has a free or blocked amino terminus.
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin; D008298:Maleates; D008722:Methods; D010196:Pancreatic Elastase; D010443:Peptide Chain Termination, Translational; D010447:Peptide Hydrolases; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000378:metabolism
D008298:Maleates; D010455:Peptides; D011506:Proteins; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D010196:Pancreatic Elastase