Novel chromatography techniques for high-throughput analysis of proteomes

Abstract

Due to their rather high complexity, most analyses of proteome samples have been accomplished by multidimensional separation techniques based on either top-down or bottom-up strategies. In the former strategy, all proteins extracted from a biological sample are separated by twodimensional gel electrophoresis (2D-GE)—introduced by O’Farrell in 1975 [1]—in which proteins are separated on a flat gel according to their isoelectric points (pIs) by isoelectric focusing (IEF) on the first dimension and molecular weights (MWs) by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) on the second dimension. Because of its unparalleled resolving power, 2D-GE has become an important technique in proteomic analysis [2]. However, it is a manual, time-consuming technique with poor sensitivity, poor quantifying power and a limited dynamic range. Moreover, it is difficult to investigate rather hydrophobic/hydrophilic proteins and those that occur with low copy number in whole cell lysate using this technique. All of these drawbacks have undermined its prospects of being the dominant separation technique in proteome research in the future. As an alternative approach, the bottom-up strategy has been paid increasing attention over the past decade. In this approach, the digest of all extracted proteins is first separated by two-dimensional high-performance liquid chromatography (2D-HPLC), and then the components are identified by tandem mass spectrometry (MS/MS) [3]. More recently, Yates and his colleagues developed a multidimensional protein identification technology (MUDPIT) that involves the 2D-HPLC separation of peptide mixtures using an orthogonal biphasic capillary column packed with strong cation exchange (SCX) resin and reversed-phase (RP) materials in tandem. They applied this novel technology to the analysis of the whole yeast proteome, and 5440 peptides (corresponding to 1484 unique proteins) were identified, among which a substantial number of low-abundance and trans-membrane proteins were identified [4]. However, the greatest impediment to the widespread use of 2D-HPLC platforms is currently the rather long analysis time required—generally about up to days per analysis. Therefore, it is crucial to accelerate the whole analysis procedure. In this article, recent advances in the development of novel chromatography techniques for the high-throughput analysis of proteomes are summarized.