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

Abstract

The vast majority of proteomic studies employ RP-HPLC coupled with MS/MS 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 studies usually require milligrams to micrograms of starting material. CZE provides an interesting alternative separation method based on a different separation mechanism than HPLC. CE 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. CZE coupled with this interface and these detectors provides single shot detection of >1250 peptides from an Escherichia coli digest in less than 1 h, identification of nearly 5000 peptides from analysis of seven fractions produced by SPE of the E. coli digest in a 6 h 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.