Microscale Reversed-Phase Liquid Chromatography/Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Deep and Highly Sensitive Bottom–Up Proteomics: Identification of 7500 Proteins with Five Micrograms of an MCF7 Proteome Digest
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Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom-up proteomics. It has approached 4000-8000 protein identifications (IDs) from a human cell line, mouse brains, or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least 500 μg of complex proteome digests were required for the LC/CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom-up proteomics of mass-limited samples. In this work, we coupled microscale reversed-phase LC (μRPLC)-based peptide prefractionation to dynamic pH-junction-based CZE-MS/MS for deep bottom-up proteomics of the MCF7 breast cancer cell proteome starting with only 5 μg of peptides. The dynamic pH-junction-based CZE enabled a 500 nL sample injection from as low as a 1.5 μL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of μRPLC prefractionation were investigated, C18 ZipTip and nanoflow RPLC. C18 ZipTip/CZE-MS/MS identified 4453 proteins from 5 μg of the MCF7 proteome digest and showed good qualitative and quantitative reproducibility. Nanoflow RPLC/CZE-MS/MS produced over 7500 protein IDs and nearly 60 000 peptide IDs from the 5 μg of MCF7 proteome digest. The nanoflow RPLC/CZE-MS/MS platform reduced the required amount of complex proteome digests for LC/CZE-MS/MS-based deep bottom-up proteomics by 2 orders of magnitude. Our work provides the proteomics community with a powerful tool for deep and highly sensitive proteomics.
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The Yang et al. results demonstrated increased sensitivity over C18 ZipTip-only analysis and highlighted advantages of an LC-CZE-MS bottom-up approach.
“These results demonstrated an increase in sensitivity over analysis after separation using C18 ZipTip only, and highlighted the advantages of using a LC-CZE-MS bottom-up approach.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [47]
Yang et al. reported a reversed-phase LC-CZE bottom-up approach for the MCF7 proteome that identified 7,500 proteins and 60,000 peptides.
“Analysis using nano reversed-phase LC combined CZE-MS resulted in the identification of 7,500 proteins and 60,000 peptides.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [47]
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Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis
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Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research
Gomes, F. P., & Yates, J. R. (2019) · Mass Spectrometry Reviews · doi:10.1002/mas.21599 · Cited by 112 · see citing works
Chen, D., Ludwig, K. R., Krokhin, O. V., Spicer, V., Yang, Z., Shen, X., Hummon, A. B., & Sun, L. (2019) · Analytical Chemistry · doi:10.1021/acs.analchem.8b04770 · Cited by 34 · see citing works
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- Authors
- Yang, Z., Shen, X., Chen, D., & Sun, L.
- Affiliations
- Michigan State University (US)
- Corresponding author
- Sun, Liangliang
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2018-08-13
- Volume
- 90
- Issue
- 17
- Pages
- 10479-10486
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC6156779
- OpenAlex
- W2887422147
- Cited by
- 43
- References
- 9
- Citations by year
- 2025: 4 · 2024: 4 · 2023: 3 · 2022: 6 · 2021: 8 · 2020: 6 · 2019: 9 · 2018: 3
- FWCI
- 3.02
- 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 (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
- shotgun proteomics, dynamic pH junction, protein identification, mass-limited samples
- MeSH terms
- Calibration, Humans, Electrophoresis, Capillary, Proteomics, Tandem Mass Spectrometry, Chromatography, Reverse-Phase, Limit of Detection, Proteolysis, MCF-7 Cells
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