A simple method for protein N-terminal confirmation by stable isotope labeling and matrix-assisted laser desorption/ionization mass spectrometry

The protein N-terminal sequence is essential for protein identification and confirmation of the removal of N-terminal methionine or signal peptides. Without special labeling, it is difficult to distinguish the protein N-terminus from lots of peptides derived by enzymatic digestion by mass spectrometry (MS). Here, a robust method based on specific d(0)/d(3)-acetylation of the protein N-terminal amino group and selective monitoring of the doublet acetylated peptide was introduced. After the protein was guanidinated, it was acetylated by a 1:1 mixture of acetic anhydride-d(0) and acetic anhydride-d(6), the derivatized protein was trypsin-digested, and monitored by matrix-assisted laser desorption/ionization (MALDI) MS. Only the amino group of the protein N-terminal peptide would be tagged by one unique d(0)/d(3)-acetyl group, which appeared as a pair of characteristic isotopic peaks with a mass difference of 3 Da in the profile of peptide mass finger-printing (PMF), whilst other internal peptides did not have such a characteristic isotopic pattern. In the next step, the recognized N-terminal peptide could be selected as a precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were successfully tested by this method and all N-terminal peptides were specially assigned. The results indicate the potential usage of this method in protein N-terminal identification.