Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
Open accessAbstract
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.
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A dynamic pH junction protocol for top-down analysis of complex samples achieved microliter-scale sample-loading volume.
“McCool and co-workers described a protocol for top-down analysis of complex samples using a dynamic pH junction where a microliter-scale sample-loading volume was achieved52.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [52]
Sample loading is the major drawback of CE and can be addressed using simple protocols, as shown in top-down and bottom-up applications.
“Sample loading is the major drawback of CE, which can be addressed using simple protocols, as demonstrated in top-down and bottom-up applications52,87.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [52]
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Self-citation
- Authors
- McCool, E. N., Lubeckyj, R., Shen, X., Kou, Q., Liu, X., & Sun, L.
- Affiliations
- Michigan State University (US)
- Indiana University – Purdue University Indianapolis (US)
- Indiana University School of Medicine (US)
- Corresponding author
- McCool, Elijah N.
- Lubeckyj, Rachele
- Venue
- Journal of Visualized Experiments
- Publisher
- MyJove Corporation
- Published
- 2018-10-24
- Issue
- 140
- ISSN
- 1940-087X (ISSN-L)
- PMC
- PMC6235596
- OpenAlex
- W2898213481
- Cited by
- 26
- References
- 34
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 3 · 2023: 3 · 2022: 1 · 2021: 3 · 2020: 2 · 2019: 10
- FWCI
- 1.62
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Visualized Experiments (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM118470, R01GM125991)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- top-down proteomics, capillary zone electrophoresis, tandem mass spectrometry, proteoforms, ion trap mass spectrometer, Escherichia coli proteome
- MeSH terms
- Electrophoresis, Capillary, Proteomics, Tandem Mass Spectrometry
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