Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface

Abstract

The vast majority of proteomic studies employ reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry for analysis of the tryptic digest of a cellular lysate. This technology is quite mature, and typically provides identification of hundreds to thousands of peptides, which is used to infer the identity of hundreds to thousands of proteins. These approaches usually require milligrams to micrograms of starting material. Capillary zone electrophoresis provides an interesting alternative separation method based on a different separation mechanism than HPLC. Capillary electrophoresis received some attention for protein analysis beginning 25 years ago. Those efforts stalled because of the limited performance of the electrospray interfaces and the limited speed and sensitivity of mass spectrometers of that era. This review considers a new electrospray interface design coupled with Orbitrap Velos and linear Q-trap mass spectrometers. Capillary zone electrophoresis coupled with this interface and these detectors provides single shot detection of >1,250 peptides from an E. coli digest in less than one hour, identification of nearly 5,000 peptides from analysis of seven fractions produced by solid-phase extraction of the E. coli digest in a six hour total analysis time, low attomole detection limits for peptides generated from standard proteins, and high zeptomole detection limits for selected ion monitoring of peptides. Incorporation of an integrated on-line immobilized trypsin microreactor allows digestion and analysis of picogram amounts of a complex eukaryotic proteome.

An Open Tubular CEC Column of Excellent Separation Efficiency for Proteomic Analysis

“…31 CEC can be employed in the 2-D separation systems, too. 16 In most of the above CEC applications in proteomics, however, either packed or monolithic columns have been used, and nano-particle based pseudostationary phases have been sometimes used.…”

Capillary zone electrophoresis for bottom‐up analysis of complex proteomes

“…These advances have been described in a number of recent reviews [29–34]. The interface of CZE with a mass spectrometer faces several technical challenges.…”

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line

“…CE-ESI-MS applications have benefited from recent improvements in the electrospray interface [5]. As a result of these improvements, CEMS has attracted renewed interest as a tool for proteomics research [6-8]. …”