Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography
Open accessAbstract
Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.
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Yu et al. used offline fractionation by high pH RPLC followed by online low pH RPLC coupled to MS to identify 2778 proteoforms from 628 proteins.
“Yu et al. took a different approach using offline fractionation by high pH RPLC followed by online low pH RPLC coupled to MS to enable the identification of 2778 proteoforms from 628 proteins [87].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [87]
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Top-down proteomics: challenges, innovations, and applications in basic and clinical research
Brown, K. A., Melby, J. A., Roberts, D. S., & Ge, Y. (2020) · Expert Review of Proteomics · doi:10.1080/14789450.2020.1855982 · Cited by 152 · see citing works
- Authors
- Yu, D., Wang, Z., Cupp-Sutton, K. A., Liu, X., & Wu, S.
- Affiliations
- University of Oklahoma (US)
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Room 2210, 73019-5251, Norman, OK, USA
- Indiana University – Purdue University Indianapolis (US)
- Corresponding author
- Wu, Si
- Venue
- Journal of the American Society for Mass Spectrometry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-11-21
- Volume
- 30
- Issue
- 12
- Pages
- 2502-2513
- ISSN
- 1044-0305 (ISSN-L)
- PMC
- PMC7539543
- OpenAlex
- W2991603986
- Cited by
- 39
- References
- 63
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 3 · 2023: 9 · 2022: 10 · 2021: 6 · 2020: 6
- FWCI
- 2.19
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Journal of the American Society for Mass Spectrometry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- National Institute of Allergy and Infectious Diseases (R01AI141625)
- National Institute of General Medical Sciences (R01GM118470)
- University of Oklahoma (FIP)
- Oklahoma Center for the Advancement of Science and Technology (HR16-125)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- top-down proteomics, proteoforms, post-translational modification, HeLa cell lysate, human cancer cells, phosphorylation, octanoylation, lipoylation, sample complexity reduction
- MeSH terms
- HeLa Cells, Humans, Hydrogen-Ion Concentration, Phosphorylation, Protein Processing, Post-Translational, Proteome, Proteomics, Tandem Mass Spectrometry, Chromatography, Reverse-Phase
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