Solution Isoelectric Focusing for Peptide Analysis:  Comparative Investigation of an Insoluble Nuclear Protein Fraction

In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, interfaced with mass spectrometry. An insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC−MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The two-dimensional separation method was combined with <sup>18</sup>O labeling for comparative analysis of protein expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the advantage that it can be accomplished after the <sup>18</sup>O labels are introduced. The labeled peptides can be mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line. Keywords: solution isoelectric focusing • shotgun proteomics • <sup>18</sup>O labeling • mass spectrometry • drug resistance • nuclear proteins • reversed-phase liquid chromatography • MCF-7 cancer cells • heterogeneous nuclear ribonucleoproteins