Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line
Open accessAbstract
Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide’s charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R 2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.
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Chen and co-workers reported a model to predict the electrophoretic mobility of phosphopeptides via CZE separation.
“More recently, Chen and co-workers reported a model to predict the electrophoretic mobility of phosphopeptides via CZE separation86.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [86]
According to modeling data, phosphorylation reduces a peptide's charge and thereby its electrophoretic mobility.
“According to this modeling data, phosphorylation reduces the charge of a peptide, thereby its electrophoretic mobility.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [86]
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Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research
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- Authors
- Chen, D., Ludwig, K. R., Krokhin, O. V., Spicer, V., Yang, Z., Shen, X., Hummon, A. B., & Sun, L.
- Affiliations
- Michigan State University (US)
- University of Notre Dame (US)
- University of Manitoba (CA)
- The Ohio State University (US)
- Corresponding author
- Sun, Liangliang
- ORCID
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-01-09
- Volume
- 91
- Issue
- 3
- Pages
- 2201-2208
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC6506858
- OpenAlex
- W2909907179
- Cited by
- 34
- References
- 50
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 3 · 2023: 2 · 2022: 6 · 2021: 5 · 2020: 5 · 2019: 8
- FWCI
- 2.40
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Division of Chemistry (CHE-1351595)
- Michigan State University
- Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-05963)
- National Institute of General Medical Sciences (R01GM110406, 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
- capillary zone electrophoresis, tandem mass spectrometry, phosphoproteomics, HCT116 cells, colon cancer, strong cation exchange, reversed-phase liquid chromatography, phosphorylation, phosphopeptide identification
- MeSH terms
- Amino Acid Sequence, Humans, Phosphopeptides, Electrophoresis, Capillary, Proteome, Proteomics, HCT116 Cells, Tandem Mass Spectrometry, Chromatography, Reverse-Phase
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