Bottom-up proteome analysis of E. coli using capillary zone electrophoresis-tandem mass spectrometry with an...

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE-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 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 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 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom-up analysis of prokaryote proteomes.