Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms
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Capillary 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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The Sun group optimized dynamic pH junction CZE using 10% acetic acid as background electrolyte to load ~1 μL and demonstrated single-shot CZE-MS identification of ~600 proteoforms from E. coli lysate.
“To address these major issues, the Sun group optimized the dynamic pH junction CZE method, using 10% acetic acid as the background electrolyte, to load ~1 μL of sample and they demonstrated the use of single-shot CZE-MS analysis to identify ~600 proteoforms from an Escherichia coli lysate [74].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [74]
A CZE separation for top-down proteome analysis of Escherichia coli resulted in a high peak capacity of about 280.
“Figure 4 shows a CZE separation for top-down proteome analysis of Escherichia coli that resulted in high peak capacity (~ 280, calculated using the average peak width at 50% peak height for the Escherichia coli proteome sample)69.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [69]
Acetic acid increased protein migration time, which may be attributed to lower pH causing increased hydrodynamic protein radii, more protein unfolding, and reduced electroosmotic flow.
“The use of acetic acid also increased the migration time of the proteins, which might be attributed to the lower pH of acetic acid that resulted in an increase in hydrodynamics in protein radii, exacerbation of protein unfolding, and reduction of EOF.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [69]
A BGE of 0.1% formic acid at pH 2.8 gave peak intensities equivalent to acetic acid BGEs.
“A BGE composed of 0.1% formic acid (pH 2.8) resulted in peak intensities equivalent to the acetic acid BGEs, as illustrated in the Figure 5.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [69]
Lubeckyj and co-workers developed a strategy for large-scale top-down analysis using Escherichia coli lysate.
“A strategy for large scale top-down analysis using lysate from Escherichia coli was developed by Lubeckyj and co-workers69.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [69]
Acetic acid BGEs at pH about 2.4 and 2.2 resulted in a wider separation window.
“Nevertheless, acetic acid BGEs (pH ~ 2.4 and ~2.2) resulted in a wider separation window.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [69]
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Identification and Quantification of Proteoforms by Mass Spectrometry
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Self-citation
- Authors
- Lubeckyj, R. A., McCool, E. N., Shen, X., Kou, Q., Liu, X., & Sun, L.
- Affiliations
- Michigan State University (US)
- Indiana University Indianapolis (US)
- Corresponding author
- Sun, Liangliang
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-11-07
- Volume
- 89
- Issue
- 22
- Pages
- 12059-12067
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5726253
- OpenAlex
- W2766426851
- Cited by
- 96
- References
- 53
- Citations by year
- 2026: 7 · 2025: 11 · 2024: 5 · 2023: 7 · 2022: 8 · 2021: 10 · 2020: 11 · 2019: 20 · 2018: 16 · 2016: 1
- FWCI
- 6.34
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- Michigan State University
- National Institute of General Medical Sciences (R01GM118470)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Keywords
- top-down proteomics, capillary zone electrophoresis, dynamic pH junction, Escherichia coli proteome, proteoforms, protein truncation, N-terminal methionine excision, signal peptide removal, post-translational modification, oxidation, acetylation, loading capacity, peak capacity
- MeSH terms
- Escherichia coli, Hydrogen-Ion Concentration, Electrophoresis, Capillary, Proteome, Spectrometry, Mass, Electrospray Ionization, Escherichia coli Proteins, Proteomics, Tandem Mass Spectrometry
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