Femtogram proteomics: We report an ultrasensitive capillary zone electrophoresis-mass spectrometry system based on an improved nanospray interface. This system is used for analysis of picogram to femtogram amounts of E. coli digests. Over 100 proteins were identified based on tandem mass spectra from 16 pg digests; over 60 proteins were identified from 400 fg digests based on accurate mass and time tags in 10 min.

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.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has fallen far below that of reversed phase liquid chromatography (RPLC)-MS/MS. Here, we report the use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (~300). This method is coupled to an Orbitrap Fusion mass spectrometer via an electro-kinetically pumped sheath flow interface for analysis of complex proteome digests. Single-shot CZE-MS/MS identified over 10 000 peptides and 2 100 proteins from a HeLa cell proteome digest in ~100 min. This performance is nearly an order of magnitude superior to earlier CZE studies and is within a factor of 2 to 4 of state-of-the-art nano ultrahigh pressure LC system.

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We report the use of the dynamic pH junction based capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) for bottom-up proteomics with an electrokinetically pumped sheath-flow nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides and produced 66% sequence coverage. In contrast, pH junction injection of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides and produced 70% coverage using a pH 6.5 sample buffer and the LTQ. A 20 nL conventional injection of a 1 mg/mL Escherichia coli digest identified 508 peptides and 199 proteins with the LTQ. A 400 nL pH junction injection of a 0.1 mg/mL E. coli digest identified 527 peptides and 179 proteins with the LTQ. Triplicate technical replicates of a 0.01 mg/mL sample with 400-nL injection volume using a pH junction identified 288 ± 9 peptides and 121 ± 5 proteins with the LTQ. There was outstanding concordance in migration time between the pH junction and normal injection. The pH junction produced narrower peaks and significant concentration for all but the most acidic components in the sample. Compared with the conventional stacking method, the pH junction method can generate comparable performance for small injection volume (20 nL) and significantly better concentration performance for a large injection volume (200 nL). We also applied the pH junction to three intact standard proteins and observed a >10× increase in peak intensity compared to conventional injection.