Proteomics based on high-efficiency capillary separations

Abstract

Identifying and quantifying in a high throughput manner the proteins expressed by cells, tissues or an organism provides the basis for understanding the functions of its constituents at a "systems" level. As a result, proteome analysis has increasingly become the focus of significant interest and research over the past decade. This is especially true following the recent stunning achievements in genomics analyses. However, unlike the static genome, the complexities and dynamism of the proteome present significant analytical challenges and demand highly efficient separations and detection technologies. A number of recent technological advancements have been in direct response to these challenges. Currently, strategically mated combinations of sophisticated separations techniques and advanced mass spectrometric detection represent the best approach to addressing the intricacies of the proteome. Liquid-phase separations, often within capillaries, are increasingly recognized as the best separations technique for this approach. In combination on-line with mass spectrometry, liquid-phase separations provide the improved analytical sensitivity, sample throughput, and quantitation capabilities necessitated by the multifaceted problems within proteomics analyses. This review focuses primarily on current high-efficiency capillary separations techniques, including both capillary liquid chromatography and capillary electrophoresis, applied to the analysis of complex proteomic samples. We emphasize developments at our laboratory and illustrate technical advances that attempt to review the role of separations within the broader context of a state-of-the-art integrated proteomics effort.

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

“…The original analytical scale size columns were supplemented with their capillary counterparts in the early 2000s [5,[12][13][14][15][16][17][18][19]. These sub-millimeter internal diameter columns facilitate direct splitless coupling with electrospray ionization mass spectrometry (ESI-MS) [20][21][22][23].…”

On the way to understand biological complexity in plants: S-nutrition as a case study for systems biology

“…Recently, in an attempt to increase throughput and to resolve proteins not easily analysed by a 2DE approach, so-called multidimensional liquid chromatography (cation exchange followed by reverse-phase column separation) coupled to ESI-MS/MS was successfully introduced [70,71]. A key challenge of proteomics in comparison to the static sequence-driven problems of genomics is that the protein complement is extremely dynamic in amount, composition, interaction and activity [72]. However, as the speed of experimental proteomic data generation increases, it appears possible to meet this challenge, but this needs to be supported with an appropriate advance in the bioinformatic tools to handle these data.…”

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

“…Among these are a small sample size requirement, high sensitivity, a wide dynamic range, and better capability for automation. 9 There are two approaches to protein identification using MS. The more frequently used one is known as the "bottom-up" approach.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] By building on these and earlier efforts, it is anticipated that the popularity of CE-MS will grow, and its advantages will be better appreciated and exploited.…”