Capillary electrophoresis applied to proteomic analysis

Abstract

In the postgenomic era, proteomics has become a dominant field for identifying and quantifying the complex protein machinery of the cell. The expression levels, post-translational modifications, and specific interactions of proteins control the biology of such processes as development, differentiation, and signal transduction. Studies of the proteins involved in these processes often leads to a better understanding of biology and of human disease. Powerful separation techniques and sensitive detection methods enable researchers to untangle these complicated networks of processes. Capillary electrophoresis coupled with either mass spectrometry or laser-induced fluorescence are two of the techniques that make this possible. This review will cover proven capillary electrophoresis-based methods for proteomics on the cell and tissue level and their application in biological and clinical studies, relevant new developments in enabling technology such as microfluidic CE-MS demonstrated on model systems, and comment on the future of CE in proteomics.

Identifying kinetically stable proteins with capillary electrophoresis

“…Because of the known correlation between SDSresistance and KS, 5,6 the SDS-PAGE boil-unboil assay was carried out to confirm the KS or lack thereof of the eight control proteins chosen for this study. As expected, the non-KSPs a-chymotrypsin (CHT), glucose dehydrogenase (GD), concanavalin A (ConA), and myoglobin (MYO) were denatured by SDS even when the samples were not heated, resulting in identical migration on the gel as the respective samples that were boiled [ Fig.…”

“…CE is also a powerful tool for protein separation because of its high efficiency, fast analysis time and low requirement for sample consumption. 6 Furthermore, CE is useful for probing the interaction between biomolecules 7 and the interaction between proteins and SDS. Thus, we applied CE to study four SDS-denatured non-KSPs as a control experiment, and the migration times for each protein were similar, consistent with the similar charge-to-mass ratio of SDS-denatured proteins.…”

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

“…CZE separates analytes by their charge-to-size ratios in buffers under a high electrical field and is often used as the final dimension prior to MS analysis, while the separation feature of CIEF is based on isoelectric point, and this technique is more suitable to be used as the first dimension separation. Detailed description of different CE-MS interfaces, sample preconcentration and capillary coating to minimize analyte adsorption could be found in several reviews (Ahmed, 2009a;Fonslow and Yates, 2009;Haselberg et al, 2007Haselberg et al, , 2011Huck et al, 2006;Klampfl, 2009;Simpson and Smith, 2005). CE technique is complementary to conventional LC in that it is suitable for the analysis of polar and chargeable compounds.…”

Proteomics Analysis of Kinetically Stable Proteins

“…In CE, proteins are typically detected using UV absorption, laser induced fluorescence (LIF), or by coupling to a mass spectrometer (MS) (Fonslow & Yates, 2009;Garcia-Campana, Taverna, & Fabre, 2007;Herrero, Ibanez, & Cifuentes, 2008;Kasicka, 2008;Stutz, 2005). A limitation when using UV detection is that the limit of detection (LOD) is in the micromolar range.…”