Single-Shot Proteomics Using Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry with Production of More than 1 250 Escherichia coli Peptide Identifications in a 50 min Separation

Abstract

Capillary zone electrophoresis (CZE)-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was optimized and applied for analysis of 1–100 ng E. coli protein digests in a single run (single-shot analysis). The system employed an electrokinetically-pumped nanospray interface, a coated capillary, and stacking conditions for sample injection. More than 1,250 peptides were identified by optimized single-shot CZE-ESI-MS/MS with 100 ng digest loaded and 50 min analysis time. When 10 ng and 1 ng digests were loaded, about 1,000 and 600 peptides were identified in a single-shot analysis, respectively. Compared with single-shot ultra-performance liquid chromatography (UPLC)-ESI-MS/MS, CZE-ESI-MS/MS produced fewer peptide IDs (1,377 ± 128 vs. 1,875 ± 32) for large sample loading amounts (100 ng) with the same mass spectrometer time (50 min). However, when the loaded digest was mass limited (1 ng), CZE-ESI-MS/MS generated many more peptide identifications than UPLC-ESI-MS/MS (627 ± 38 vs. 342 ± 113). In addition, CZE-ESI-MS/MS and UPLC-ESI -MS/MS provided complementary peptide level identifications. These results suggest that CZE-ESI-MS/MS may be useful for large-scale, comprehensive, and confident proteomics analysis.

Advances in microscale separations towards nanoproteomics applications

“…A single shot CE-MS analysis of 400 ng of HeLa cell lysate digests has resulted in identification of ~10,000 peptides and 2100 proteins, which is approximately 2.5-fold lower than the number from nanoLC-MS using a 300 ng sample. There is 70% overlap of the identified peptides between the CE and LC methods, but CE tends to identify larger peptides than LC [57,58]. More recently, a number of groups developed sheathless CE-MS interfaces where the electroosmotic flow from capillary is the only source of nanospray [49,51].…”

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

“…As FASS can be effective just by using a low σ sample, it has been mostly employed among all the OSP methods in CE-MS. One of the most popular applications is proteome analysis [41][42][43][44]. Dovichi's group published several papers on FASS-CE-nanoESI-MS for high performance bottom-up proteome analysis [41,42].…”

“…Dovichi's group published several papers on FASS-CE-nanoESI-MS for high performance bottom-up proteome analysis [41,42]. They identify 167 proteins from 1 ng E. coli proteins and 100 phosphorylated proteins from only 2 ng proteins from MCF-10A cell line.…”

Developments in Interfacing Designs for CE–MS: Towards Enabling Tools for Proteomics and Metabolomics

“…Since the latest update on this topic [53], a few new studies have appeared [65][66][67][68]. All these studies are from the group of Dovichi and, therefore, make use of the EOF driven sheath-liquid interface ("Advancements in CE-MS Interface Designs").…”

“…Up to 600 peptides, corresponding to 160 proteins, could be identified in this study (using 84 ng digest injections). This number could be improved by making use of a neutrally-coated capillary as it provides higher separation efficiencies and more time to obtain tandem mass spectra, resulting in the identification of over 1,250 peptides from an injection of 100 ng of E. coli digest in a single-shot approach [66]. This was just slightly lower than using UPLC-MS/MS and, interestingly, complementary peptide identifications were obtained with both techniques.…”