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Combining microscale solution‐phase isoelectric focusing with Multiplexed Proteomics® dye staining to analyze protein post‐translational modifications
Author(s) -
Schulenberg Birte,
Patton Wayne F.
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.200406009
Subject(s) - isoelectric focusing , proteome , proteomics , gel electrophoresis , chromatography , staining , immobilized ph gradient , polyacrylamide gel electrophoresis , difference gel electrophoresis , chemistry , lysis buffer , biochemistry , microbiology and biotechnology , biology , lysis , genetics , gene , enzyme
Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution‐phase isoelectric focusing is an effective alternative to sucrose‐gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence‐based multiplexing dye technologies greatly extend the capacity of SDS‐polyacrylamide gel electrophoresis with respect to the investigation of proteome‐wide changes in protein expression and post‐translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics® staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two‐dimensional gel electrophoresis.