Bottom-up proteome analysis of E. coli using capillary zone electrophoresis-tandem mass spectrometry with an electrokinetic sheath-flow electrospray interface

摘要

The E. coli proteome was digested with trypsin and fractionated using solid phase extraction on a C18 SPE column. Seven fractions were collected and analyzed by capillary zone electrophoresis (CZE)-electrospray ionization tandem mass spectrometry (ESI-MS/MS). The separation was performed in a 60 cm long linear polyacrylamide-coated capillary with a 0.1% (v/v) formic acid separation buffer. An electrokinetic sheath-flow electrospray interface was used to couple the separation capillary with an Orbitrap Velos operating in higher-energy collisional dissociation mode. Each CZE-ESI-MS/MS run lasted 50 minutes and total MS time was 350 minutes. A total of 23,706 peptide spectra matches, 4,902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than six hours, the sample identification rate (145 proteins/hour) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly four-fold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom-up analysis of prokaryote proteomes.