Detachable Strong Cation Exchange Monolith, Integrated with Capillary Zone Electrophoresis and Coupled with pH Gradient Elution, Produces Improved Sensitivity and Numbers of Peptide Identifications during Bottom-up Analysis of Complex Proteomes
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A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.
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CZE-MS and LC-MS identified no significant difference in the number of peptides and proteins for E. coli samples using on-line SPE preconcentration with dynamic pH separation.
“There was no significant difference in the number of peptides and proteins identified by CZE-MS and LC-MS methods.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [125]
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- Authors
- Zhang, Z., Yan, X., Sun, L., Zhu, G., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2015-04-10
- Volume
- 87
- Issue
- 8
- Pages
- 4572-4577
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC4406858
- OpenAlex
- W2330249723
- Cited by
- 67
- References
- 30
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 4 · 2023: 2 · 2022: 2 · 2021: 5 · 2020: 5 · 2019: 3 · 2018: 11 · 2017: 8 · 2016: 14 · 2015: 7 · 2013: 1
- FWCI
- 5.95
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (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, pH gradient elution, solid-phase extraction, shotgun proteomics, peptide identification, sample fractionation, dynamic pH junction, sample stacking, low-abundance peptides, Escherichia coli, tryptic peptides
- MeSH terms
- Cations, Hydrogen-Ion Concentration, Peptides, Silicon Dioxide, Sulfonic Acids, Electrophoresis, Capillary, Proteome, Solid Phase Extraction, Tandem Mass Spectrometry
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Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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