Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry for Proteomic Analysis

Abstract

We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) CE-MS/MS procedure which employs a high sensitivity porous ESI sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.

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

“…Similar complementary peptide identification was found in recent CE and nano-LC studies by our group and other researchers (32,39,58). CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass.…”

“…CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass. Additionally, the Yates Lab attributed gains in sensitivity to lower noise levels with CE, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides relative to LC (58). These trends may be also explained by the difference in electrospray conditions between the methods.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…However, since only the porous end of the separation capillary is housed in the reservoir, the passage of ions from the reservoir through the pores in the separation capillary is achieved without dilution of the BGE. A number of groups have utilized this interface in proteomics experiments [1, [66][67][68][69][70]. The interface is sold commercially via SCIEX (Toronto, Canada) and marketed for CZE-MS applications as a part of SCIEX's CESI platform.…”

Recent Studies on Online Sample Preconcentration Methods inCapillary Electrophoresis Coupled with Mass Spectrometry

“…As with FASS and PHJ, major application field of tITP-CE-MS includes proteomics [82][83][84][85][86][87][88]. Wang et al reported combination of reverse-phase solid phase microextraction with tITP-CZE-nanoESI-MS [82].…”

“…Wang et al reported combination of reverse-phase solid phase microextraction with tITP-CZE-nanoESI-MS [82]. They succeeded in identification of up to 370 proteins from 50 ng of Xenopus laevis proteins.…”