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Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples

Authors
  • Lubeckyj, Rachele A.1
  • Basharat, Abdul Rehman2
  • Shen, Xiaojing1
  • Liu, Xiaowen2, 3
  • Sun, Liangliang1
  • 1 Michigan State University, 578 S Shaw Ln, East Lansing, MI, 48824, USA , East Lansing (United States)
  • 2 Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202, USA , Indianapolis (United States)
  • 3 Indiana University School of Medicine, Indianapolis, IN, 46202, USA , Indianapolis (United States)
Type
Published Article
Journal
Journal of the American Society for Mass Spectrometry
Publisher
Springer-Verlag
Publication Date
Apr 09, 2019
Volume
30
Issue
8
Pages
1435–1445
Identifiers
DOI: 10.1007/s13361-019-02167-w
Source
Springer Nature
Keywords
License
Yellow

Abstract

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention recently for top-down proteomics because it can achieve highly efficient separation and very sensitive detection of proteins. However, separation window and sample loading volume of CZE need to be boosted for a better proteome coverage using CZE-MS/MS. Here, we present an improved CZE-MS/MS system that achieved a 180-min separation window and a 2-μL sample loading volume for top-down characterization of protein mixtures. The system obtained highly efficient separation of proteins with nearly one million theoretical plates for myoglobin and enabled baseline separation of three different proteoforms of myoglobin. The CZE-MS/MS system identified 797 ± 21 proteoforms and 258 ± 7 proteins (n = 2) from an Escherichia coli (E. coli) proteome sample in a single run with only 250 ng of proteins injected. The system still identified 449 ± 40 proteoforms and 173 ± 6 proteins (n = 2) from the E. coli sample when only 25 ng of proteins were injected per run. Single-shot CZE-MS/MS analyses of zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions identified 1730 ± 196 proteoforms (n = 3) and 2024 ± 255 proteoforms (n = 3), respectively, with only 500-ng proteins loaded per run. Label-free quantitative top-down proteomics of zebrafish brain Cb and Teo regions revealed significant differences between Cb and Teo regarding the proteoform abundance. Over 700 proteoforms from 131 proteins had significantly higher abundance in Cb compared to Teo, and these proteins were highly enriched in several biological processes, including muscle contraction, glycolytic process, and mesenchyme migration. Graphical Abstract

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