Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.
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.
Janini
George M
GM
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute, Frederick, MD, USA.
Chan
King C
KC
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute, Frederick, MD, USA.
Conrads
Thomas P
TP
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute, Frederick, MD, USA.
Issaq
Haleem J
HJ
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute, Frederick, MD, USA.
Veenstra
Timothy D
TD
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute, Frederick, MD, USA.
D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D010455:Peptides / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D001798:Blood Proteins; D010455:Peptides