CZE and CIEF of proteins have preceded, and accompanied, the birth of proteomics. Although they might not be fully exploited in massive proteomic analyses (especially those projects aiming at a deep discovery of possibly the entire proteome of a cell or subcellular organelles or biological fluids), it still has interesting features and advantages, especially with samples of limited heterogeneity and in the field of purity checking for recombinant DNA proteins meant for human consumption. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to analysis of proteins and peptides, while commenting on the constraints and caveats of the technique. The tutorial ends with an outlook on the future, which might be dominated by microchip electrophoresis, especially for ultrafast analyses of protein samples in a sieving mode, in presence of either sieving liquid polymers or firm gels polymerized within the microchannels. To this purpose, commercial instrumentation is already available on the market. This tutorial is part of the International Proteomics Tutorial Programme (IPTP 13).

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N ␣ -terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example. Molecular & Cellular

Two approaches were used to prepare a series of surface-modified capillaries. In the first, a sublayer was formed by coupling gamma-methacryloxypropyltrimethoxysilane to the surface silanol groups forming an SI-O bond; a top layer was then formed by polymerizing acrylamide in the capillary, which reacted with the sublayer. In the second approach, a sublayer was formed by silanol chlorination, followed by Grignard coupling of vinylmagnesium bromide to form an Si-C bond at the surface; a top layer was formed by polymerizing either acrylamide (AA), dimethylacrylamide (DMA), N-acryloylaminoethoxyethanol (AAEE), or N-acryloylaminopropanol (AAP) onto the sublayer. The Si-Cpoly(AA) capillaries were more stable and produced an approximately 10-fold lower electroosmotic flow compared to the Si-O-poly(AA) capillaries. The Si-C sublayer was used to compare the performance of all four top layers. Electroosmotic flow decreased in the order: Si-O-poly(AA), Si-C-poly(AA), Si-Cpoly(AAEE), Si-C-poly(DMA), and Si-C-poly(AAP). Si-C-poly(AA) showed evidence of irreversible degradation at pH 9 already after 40-50 runs. Si-C-polyAAP-coated capillaries demonstrated superior efficiency and migration time reproducibility for a number of alkaline proteins and for fluorescently labeled ovalbumin. Excellent performance was maintained, in the case of poly(AAP), for a least 300 runs (of 30 min duration) at pH 9.0.

High-resolution capillary zone electrophoresis – mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2. Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography – mass spectrometry-based approaches.

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