Electrophoretic mobility-assisted identification of proteins by nanoelectrospray capillary electrophoresis/mass spectrometry under methanolic conditions
H. Katayama et al., Electrophoretic mobility-assisted identification of proteins by nanoelectrospray capillary electrophoresis/mass spectrometry under methanolic conditions, RAP C MASS, 14(14), 2000, pp. 1167-1178
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.