Optimization and Evaluation of a Sheathless Capillary Electrophoresis–Electrospray Ionization Mass Spectrometry Platform for Peptide Analysis: Comparison to Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Abstract

In this study we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) interface with a porous tip as the nanospray emitter for use in peptide analysis. A positively charged capillary coating and 0.1% formic acid as background electrolyte were used for separation upstream from mass spectrometry characterization. The influence of the distance between emitter tip and MS inlet, ESI voltage applied, and of the electroosmotic flow (EOF) on electrospray performance and efficiency of the system was investigated in detail. Under optimized conditions, less than 30 amol of a model peptide (angiotensin I) was required for a detection in the base peak electropherogram and positive identification via tandem MS. Three different cationic capillary coatings were investigated for stability, resolution, and EOF and were found to enable reproducible separations by CE-ESI-MS. After optimizing MS settings, the effectiveness of the CE-ESI-MS method developed was compared with a state-of-the-art nano-liquid chromatography (LC)-ESI-MS method by analyzing Arg-C-digested rat testis linker histones with both systems. With comparable amounts of sample applied, the number of identified peptides increased by more than 60% when using CE-ESI-MS. We found that low molecular mass peptides (below 1400 Da) were preferentially identified by CE-ESI-MS, since this group of peptides poorly interacted with the reversed-phase material in the nano-LC system. Finally, total analysis time in LC-ESI-MS for three runs including equilibration was nearly 4 times longer than that of CE-ESI-MS: 246 versus 66 min.

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

“…Some of the larger phosphopeptides, however, were preferentially detected in LC-MS. Our group recently described this effect for unmodified H1 peptides. We found that low-molecular-mass peptides (below 1400 Da) were preferentially identified by CESI-MS, because these peptides interact poorly with the RP material in the nano-LC system and therefore are washed out (39).…”

“…2A shows the base peak electropherogram of 300 fmol histone H1 digest obtained with the PEI coated capillary, 0.1% formic acid as a BGE, and a separation voltage of Ϫ25 kV. Because of the strong positively charged coating, a rather high electroosmotic flow rate of ϳ120 to 135 nl/min was generated, resulting in very short migration times (39). The sample was run in triplicate, and MS/MS spectra obtained were searched against the International Protein Index rat database.…”

“…These interact poorly with the RP material and elute in the void volume, so they cannot be detected via LC-ESI-MS (39). This effect should be even more pronounced with small and hydrophilic phosphopeptides.…”

“…Our group recently described important features of CESI-MS and reported the comparison of this method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis consisting mainly of different histone H1 subtypes (39). The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides.…”

“…The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides. The CESI-MS method provided shorter analysis times, narrower peaks yielding high signals, and the identification of a greater number of low molecular mass range peptides than LC-ESI-MS (39).…”