Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

Abstract

We have reported a set of electrokinetically pumped sheath flow nanoelectrospray interfaces to couple capillary zone electrophoresis with mass spectrometry. A separation capillary is threaded through a cross into a glass emitter. A side arm provides fluidic contact with a sheath buffer reservoir that is connected to a power-supply. The potential applied to the sheath buffer drives electro-osmosis in the emitter to pump the sheath fluid at nanoliter/minute rates. Our first generation interface placed a flat-tipped capillary in the emitter. Sensitivity was inversely related to orifice size and to the distance from the capillary tip to the emitter orifice. A second generation interface used a capillary with an etched tip that allowed the capillary exit to approach within a few hundred micrometers of the emitter orifice, resulting in a significant increase in sensitivity. In both the first and second-generation interfaces, the emitter diameter was typically 8-μm; these narrow orifices were susceptible to plugging and tended to have limited lifetime. We now report a third-generation interface that employs a larger diameter emitter orifice with very short distance between the capillary tip and the emitter orifice. This modified interface is much more robust and produces much longer lifetime than our earlier designs with no loss in sensitivity. We evaluated the third-generation interface for a 5,000-min (127 runs, 3.5 days) repetitive analysis of bovine serum albumin digest using an uncoated capillary. We observed a 10% relative standard deviation in peak area, an average of 160,000 theoretical plates, and very low carry-over (much less than 1%). We employed a linear-polyacrylamide (LPA) coated capillary for single-shot, bottom-up proteomic analysis of 300 ng of Xenopus laevis fertilized egg proteome digest, and identified 1,249 protein groups and 4,038 peptides in a 110 min separation using an LTQ-Orbitrap Velos mass spectrometer; peak capacity was ~330. The proteome dataset using this third generation interface based CZE-MS/MS is similar in size to that generated using a commercial ultra-performance liquid chromatographic analysis of the same sample with the same mass spectrometer and similar analysis time.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

“…To assess proteins at trace levels, various CE approaches can help enrich molecules on-column and separate them in increased peak capacity (64). New-generation interfaces that minimize/ eliminate sample dilution between CE and ESI may be used to ionize peptides more efficiently (see reviews in Ref (33,(65)(66)(67)); electrokinetically pumped sheath-flow (32,38) and sheathless (68,69) interfaces are promising designs in this direction. Based on recent successes in proteome coverage and post-translational analysis by CE (35,37,69), we expect these innovative solutions combined with new-generation FIG.…”

“…A microfluidic setting extended these electrophoretic studies to higher throughput, 12 erythrocytes/min (24). Continuous developments in CE separation (29,30) and lategeneration CE nano-flow ESI interfaces (31,32) (see reviews (33,34)) accomplished high-sensitivity detection of proteomes (35,36) and labile PTMs, such as phosphorylation (37). These developments enabled femtogram (zeptomole) limit of detection (38) for protein digests from cell populations.…”

Single‐Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16‐Cell Frog (Xenopus) Embryo

“…HRMS-based detection of globulins was accelerated to 12 erythrocytes/min using microfluidic devices [5] and, most recently,3 4t argeted proteins contributing to hematopoiesis were detected at % 1000 cell/s throughput using mass cytometry. [6] Continuous developments in HRMS sensitivity enabled untargeted (discovery) proteomics on progressively smaller populations of cells [1c, 7] with representative successes including the identification of 2000 proteins from 2000-4000 cells from single Langerhans islands, [8] 167 proteins from 500 breast cancer cells, [1a] 109 proteins from 100 HeLa cells, [9] and most recently, % 12 000 proteins in % 50 fertilized [10] and as ingle unfertilized egg of the South African clawed frog (Xenopus laevis). [11] Using advanced microsampling and discovery HRMS, [1c, 12] neuropeptides were characterized in single molluscan [13] and arthropod neurons [12] as well as cells in the mammalian pituitary gland and Langerhans islets.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Yet another sheath-flow interface has been reported capable of delivering coaxial sheath-liquid flow rates in the nL/min range, thereby resulting in significantly less dilution at the CZE-ESI-MS junction [61,62]. Instead of using pressure to generate coaxial sheath-liquid flow, the sheath-liquid was supplied to the junction electrokinetically, which mitigates dilution effects at the interface between CZE and ESI-MS. Reports with this setup have shown performance comparable to LC-based platforms [63].…”