Single-Shot Capillary Zone Electrophoresis–Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample
Open accessAbstract
The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.
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- [30]Zhang Z; Yan X; Sun L; Zhu G; Dovichi NJ Integrated strong cation-exchange hybrid monolith coupled with capillary zone electrophoresis and simultaneous dynamic pH junction for large-volume proteomic analysis by mass spectrometry. Anal. Chem 2015, 87, 4572– 4577. 25863379 PMC4394190
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- Authors
- Zhang, Z., Hebert, A. S., Westphall, M. S., Coon, J. J., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- University of Wisconsin–Madison (US)
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-06-10
- Volume
- 18
- Issue
- 8
- Pages
- 3166-3173
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- PMC
- PMC6679793
- OpenAlex
- W2951047141
- Cited by
- 22
- References
- 54
- Citations by year
- 2025: 1 · 2024: 3 · 2023: 3 · 2022: 3 · 2021: 6 · 2020: 3 · 2019: 3
- FWCI
- 1.50
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Journal of Proteome Research (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (R01HD084399, R35GM118110, P41GM108538, R01GM096767)
- 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
- capillary zone electrophoresis, phosphoproteomics, phosphopeptide identification, Orbitrap Fusion Lumos, electroosmotic flow, mouse brain, post-translational modification, migration time
- MeSH terms
- Algorithms, Chromatography, Liquid, Phosphopeptides, Electrophoresis, Capillary, Spectrometry, Mass, Electrospray Ionization, Escherichia coli Proteins, Proteomics, Tandem Mass Spectrometry
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