ac7b02532_si_002.xlsx (228.67 kB)
Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms
dataset
posted on 2017-10-24, 00:00 authored by Rachele
A. Lubeckyj, Elijah N. McCool, Xiaojing Shen, Qiang Kou, Xiaowen Liu, Liangliang SunCapillary zone electrophoresis-electrospray
ionization-tandem mass
spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable
platform for top-down proteomics. However, the scale of top-down proteomics
using CZE-MS/MS is still limited due to the low loading capacity and
narrow separation window of CZE. In this work, for the first time
we systematically evaluated the dynamic pH junction method for focusing
of intact proteins during CZE-MS. The optimized dynamic pH junction-based
CZE-MS/MS approached a 1 μL loading capacity, 90 min separation
window, and high peak capacity (∼280) for characterization
of an Escherichia coli proteome. The
results represent the largest loading capacity and the highest peak
capacity of CZE for top-down characterization of complex proteomes.
Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches,
nearly 600 proteoforms, and 200 proteins from the Escherichia
coli proteome with spectrum-level false discovery
rate (FDR) less than 1%. The number of identified proteoforms in this
work is over three times higher than that in previous single-shot
CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal
peptide removal, and some post-translational modifications including
oxidation and acetylation were detected.
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pH junction method1 μ L loading capacityCZE-MSFDR600 Escherichia coli Proteoforms Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometryEscherichia coli proteomecharacterizationSingle-Shot Top-Down Proteomicsloading capacityN-terminal methionine excisionCZE-ESI-MSsignal peptide removalCapillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometryprotein90 min separation windowproteoformtop-down proteomicsCZE
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