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
journal contributionposted on 2018-09-04, 00:00 authored by Zhenbin Zhang, Alexander S. Hebert, Michael S. Westphall, Yanyan Qu, Joshua J. Coon, Norman J. Dovichi
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.
120 mininjection timeloading amount2 timesprotein identificationssheath-flow nanoelectrospray interface27 000Orbitrap Fusion Lumos Tribrid Mass Spectrometerisolation window4400 Protein IdentificationsAdvanced-Peak-Determination AlgorithmThird-Generation Electrokinetically-Pumped Sheath-Flow Nanospray InterfaceIDs220 ngCZE-ESI-MSOrbitrap Fusion Lumos Tribrid platform2.7 timesseparation capillary27 000 peptide identifications27 000 PeptideVery-Low-Electroosmosis Coated CapillaryAPD algorithmK 562-cellhigh-sensitivity ionization4400 protein-group identificationspeak algorithm