Micro-proteome analysis using micro-chromatofocusing in intact protein separations

Multi-dimensional liquid-based separation is required for fractionation and mapping of complex protein mixtures from cells. A method that has been used as the first dimension in such separations is chromatofocusing (CF), which is based on generating a pH gradient on an anion exchange column. The use of pH in the first dimension is essential where pH is a fundamental property of proteins and can provide information on post-translationally modified forms of a protein. In this work, a micro-chromatofocusing technique is introduced which can separate microgram levels of proteins from cell lysates for further analysis by LC–MS/MS. It is shown that this method can analyze 10 μg of sample and detect nearly 700–800 proteins from ovarian cancer cell line lysates.