Electrophoretic mobility-assisted identification of proteins by nanoelectrospray capillary electrophoresis/mass spectrometry under methanolic conditions

A novel method for electrophoretic mobility-assisted identifications of pro teins, using capillary electrophoresis/mass spectrometry (CE/MS) under meth anolic conditions, was developed. The number of functional groups of the en zymatic digest peptides was estimated from a single run CE/MS analysis and utilized as an additional tag for database searching in addition to the mas s map of the peptides, The additional amino acid information thus obtained can improve the confidence level of the protein identification. The databas e searching software algorithm Profound was modified to accept the tag, bas ed on this new concept. In this study, optimization of the CE/MS conditions for the estimation of basic functional groups was performed as an. example . An accurate value of the number of such functional groups was obtained fr om CE characteristics when methanolic buffer (methanol/formic acid/water = 60:20:20) was used, via an excellent correlation (r = 0.997) between the nu mber of functional groups of the peptides and [MW(2/3)]. The mass spectrome try sensitivity was also improved when using the methanolic buffer in compa rison with that obtained using aqueous 1% formic acid buffer. The identific ation of a protein of Saccharomyces cerevisiae, which was separated by two- dimensional electrophoresis, was performed using the methanolic buffer in c ombination with sheathless nanoelectrospray CE/MS, A protein spot that had not been identified by MALDI-TOFMS and LC/MS/MS was successfully identified using this new method. Copyright (C) 2000 John Wiley & Sons, Ltd.