Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications.
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.
Schulenberg
Birte
B
Proteomics Department, Molecular Probes, Inc., Eugene, OR 97402, USA. birte.schulenberg@probes.com
Patton
Wayne F
WF
D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D008929:Mitochondria, Heart / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D010750:Phosphoproteins