An evaluation of the utility of in vacuo methylation for mass-spectrometry-based analyses of peptides.

In vacuo trimethylation of the N-terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI-MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N-terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N-terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side-chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI-MS signal improvement. The experimental results demonstrate that the N-terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine-containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed.