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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.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC6508074 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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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.

Zhang Zhenbin Z Hebert Alexander S AS Westphall Michael S MS Qu Yanyan Y Coon Joshua J JJ Dovichi Norman J NJ

Analytical chemistry 20180924 20

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 ( ...[more]

PMID: 30179504

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Project description: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 per 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 ?m; 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 previous designs with no loss in sensitivity. We evaluated the third-generation interface for a 5000 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 1249 protein groups and 4038 peptides in a 110 min separation using an LTQ-Orbitrap Velos mass spectrometer; peak capacity was ?330. The proteome data set using this third-generation interface-based CZE-MS/MS is similar in size to that generated using a commercial ultraperformance liquid chromatographic analysis of the same sample with the same mass spectrometer and similar analysis time.

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