Letter: Specific Isotope Labeling for the Identification of Free N-Terminal Peptides of Proteins Separated by Polyacrylamide Gel Electrophoresis

Recently, we reported a method for the identification of free or blocked N-terminal peptides of proteins. 1 Briefly, the amino groups on lysine side chains and the N-terminal of the gel-entrapped protein are blocked with normal acetic or succinic anhydride and digested with a high-specificity protease. The generated peptides are retreated with an equimolar mixture of normal and deuterated acetic anhydride. In the mass spectrum, all the peptides show a complex isotopic ion distribution due to the artificial incorporation of heavy isotopes, with the exception of the N-terminal peptide that appears as a normal signal, allowing its rapid identification. Two main problems were observed in this method: (1) during electrospray ionization mass spectrometry (ESIMS) analysis, the C-terminal ions of any internal peptide originated by an in-source fragmentation process also have natural isotopic ion distribution and could be missassigned as an N-terminal peptide and (2) since the N-terminal peptide is not isotopically-labeled, their N- and the C-terminal ions can not be differentiated in the tandem mass spectrum (MS/MS) to achieve an easier and more reliable sequencing which is particularly advantageous when proteins derived from an organism with an unknown genome are analyzed. In this sense, a number of methods have been developed to facilitate the interpretation of MS/MS spectra. 2–7 In some of these works, a stable isotope label is introduced at the peptide N-terminus using a 1 : 1 mixture of normal and deuterated acetic anhydride, allowing an easy and unambiguous identification of the N- and C-terminal ion series in the product ion spectrum. 8,9