Over 2300 Phosphorylated Peptide Identifications with Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry in a 100 min Separation
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Ultraperformance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE)-ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 μg vs 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3313 vs 1783). However, when the same sample loading amounts were used for CZE and UPLC (2-200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from a 2 μg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from a 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over 1 order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS.
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Ludwig and co-workers reported a CZE-MS approach that identified over 2,300 phosphorylated peptides in a single analysis, illustrating advantages of CZE over LC for PTM analysis.
“Ludwig and co-workers reported a CZE-MS approach that was able to identify over 2,300 phosphorylated peptides in a single analysis85, thus illustrating the advantages of CZE separation over LC for PTM analysis.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [85]
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SPE-CZE-MS quantifies zeptomole concentrations of phosphorylated peptidesPreprint
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- Authors
- Ludwig, K. R., Sun, L., Zhu, G., Dovichi, N. J., & Hummon, A. B.
- Affiliations
- University of Notre Dame (US)
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
- Corresponding author
- Hummon, Amanda B.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2015-09-25
- Volume
- 87
- Issue
- 19
- Pages
- 9532-9537
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC4605816
- OpenAlex
- W2298101292
- Cited by
- 56
- References
- 34
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 4 · 2023: 1 · 2022: 3 · 2021: 3 · 2020: 1 · 2019: 8 · 2018: 9 · 2017: 10 · 2016: 9 · 2015: 2 · 2013: 1
- FWCI
- 4.45
- 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)
- Funders
- Division of Chemistry (CHE-1351595)
- National Institutes of Health (R01GM096767, R01GM110406)
- 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, sample loading capacity, dynamic pH junction, MCF10A cells
- MeSH terms
- Chromatography, High Pressure Liquid, Humans, Peptides, Phosphorylation, Electrophoresis, Capillary, Cell Line, Tumor, Tandem Mass Spectrometry
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