Production of Over 27 000 Peptide and Nearly 4400 Protein Identifications by Single-Shot Capillary-Zone Electrophoresis–Mass Spectrometry via Combination of a Very-Low-Electroosmosis Coated Capillary, a Third-Generation Electrokinetically-Pumped Sheath-Flow Nanospray Interface, an Orbitrap Fusion Lumos Tribrid Mass Spectrometer, and an Advanced-Peak-Determination Algorithm

Abstract

We show that capillary-zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) generates very large numbers of peptide and protein identifications (IDs) by combining four technologies: a separation capillary coated to generate very low electroosmosis, an electrokinetically pumped sheath-flow nanoelectrospray interface to produce high-sensitivity ionization, an Orbitrap Fusion Lumos Tribrid platform to provide high-speed analysis, and an advanced-peak-determination (APD) algorithm to take advantage of the mass spectrometer's data-acquisition speed. The use of the APD algorithm resulted in 2 times more identifications than the standard peak algorithm. We also investigated the effect of the isolation window, injection time, and loading amount. Optimization of these parameters produced over 27 000 peptide identifications and nearly 4400 protein-group identifications from 220 ng of K562-cell digest in a single 120 min run, which is 2.7 times more IDs produced by CZE-ESI-MS/MS than by the previous state-of-the-art technique.

Single-cell Proteomics: Progress and Prospects

“…These ordered separations reduce dispersion and provide highly efficient separations at much lower operating pressures than a typical nanoLC column. They also promise improved robustness with (57). CE has been used extensively for single-cell proteomics, but so far the cells analyzed have generally been oocytes and blastocysts, etc.…”

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows

“…In addition, due to the high reactivity of the label, these excess materials are often considered unsuitable for use following rehydration and are typically discarded as waste. 11 Alternative approaches to NanoLC are gaining in popularity, with multiple studies using capillary zone electrophoresis 14,15 and microflow chromatography 16,17 demonstrating promise as alternative methodologies. Recent work has also described the use of "standard flow" proteomics, reaching the conclusion that flow rates in the 50 -200 µl per minute range can provide quality proteomics data with proper optimization.…”

Recent Advances and New Perspectives in Capillary Electrophoresis-Mass Spectrometry for Single Cell “Omics”

“…Many other groups have made strides to improve analyte identification capabilities using CE-MS. The Dovichi group improved the number of peptide and protein identifications through CZE-ESI-MS/MS by 2.7-fold more than the previous record for single-shot analysis [64]. The previous record was 10,274 peptide IDs from 440 ng of HeLa digest, and the Dovichi group was able to generate over 27,000 peptide IDs from 220 ng of human K562 protein digest [64].…”

“…The Dovichi group improved the number of peptide and protein identifications through CZE-ESI-MS/MS by 2.7-fold more than the previous record for single-shot analysis [64]. The previous record was 10,274 peptide IDs from 440 ng of HeLa digest, and the Dovichi group was able to generate over 27,000 peptide IDs from 220 ng of human K562 protein digest [64]. Using reverse phase (RP)-HPLC fractionation and sequential injections into a CE-MS system, the Lindner group was able to identify 62,000 peptides and more than 6100 proteins within 12.5 h, which is the highest number of peptides obtained for the analysis of a single proteome using CE-MS to date [65].…”