Letter: Specific isotope labeling for the identification of free N-terminal peptide of proteins separated by polyacrylamide gel electrophoresis.

We report here a method for the identification of free N-terminal peptide of in gel digested isolated proteins. It is based in the difference between the isotopic ion distribution of N-terminal peptide and internal peptides. After guanidination of lysine residues, the primary amino groups of the gel-entrapped protein are blocked with an equimolar mixture of normal and deuterated acetic anhydride. Upon MS analysis internal peptides display a normal isotopic ion distribution while the N-terminal peptide shows a complex isotopic ion distribution.

Double acylation for identification of amino-terminal peptides of proteins isolated by polyacrylamide gel electrophoresis.

We report here a method for the identification of free or blocked N-terminal peptide of in-gel digested isolated proteins. The primary amino groups of the gel-entrapped protein are blocked with normal acetic or succinic anhydride, and the protein is digested with a high-specificity protease. The generated peptides are treated with an equimolar mixture of normal and deuterated acetic anhydride. Upon mass spectrometric analysis internal peptides display a complex isotopic ion distribution while the N-terminal peptide shows a normal isotopic ion distribution. The procedure was applied to the identification of the N-terminus of individual and protein mixtures isolated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).