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Two‐dimensional liquid chromatography‐capillary zone electrophoresis‐sheathless electrospray ionization‐mass spectrometry: Evaluation for peptide analysis and protein identification
Author(s) -
Janini George M.,
Chan King C.,
Conrads Thomas P.,
Issaq Haleem J.,
Veenstra Timothy D.
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200405948
Subject(s) - chromatography , chemistry , mass spectrometry , electrospray , electrospray ionization , capillary electrophoresis , capillary electrophoresis–mass spectrometry , analytical chemistry (journal) , tandem mass spectrometry , analyte , resolution (logic) , electrophoresis , artificial intelligence , computer science
Abstract A peptide separation strategy that combines two‐dimensional (2‐D) liquid chromatography (LC)‐capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high‐abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed‐phase (RP)‐LC. Compared to ion‐exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off‐line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure‐assisted flow. Unlike first‐dimensional ion‐exchange LC fractionation, where there is a large degree of overlap, the CZE‐MS results show less than 15% overlap between neighboring RPLC fractions.