A Multidimensional Chromatography Technology for In-depth Phosphoproteome Analysis
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Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.
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S27 and S29 of Rad26 are phosphorylated in a Mec1-dependent manner.
“a proteome-wide study of protein phosphorylation reported that S27 and S29 of Rad26 are phosphorylated in a Mec1-dependent manner (4).”
From A Role for Checkpoint Kinase-Dependent Rad26 Phosphorylation in Transcription-Coupled DNA Repair in <i>Saccharomyces cerevisiae</i> (2010) · cites this paper as [4]
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- Authors
- Albuquerque, C. P., Smolka, M. B., Payne, S. H., Bafna, V., Eng, J., & Zhou, H.
- Affiliations
- University of California San Diego (US)
- Institute for Systems Biology (US)
- Corresponding author
- Zhou, Huilin
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2008-07
- Volume
- 7
- Issue
- 7
- Pages
- 1389-1396
- ISSN
- 1535-9476 (ISSN-L)
- PMC
- PMC2493382
- OpenAlex
- W2123929534
- Cited by
- 504
- References
- 32
- Citations by year
- 2026: 9 · 2025: 7 · 2024: 10 · 2023: 18 · 2022: 30 · 2021: 28 · 2020: 31 · 2019: 37 · 2018: 34 · 2017: 31 · 2016: 25 · 2015: 28 · 2014: 27 · 2013: 28 · 2012: 49
- FWCI
- 16.15
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Science Foundation (0504645)
- Ludwig Institute for Cancer Research (GM080469)
- National Institutes of Health
- National Institute of General Medical Sciences (GM 080469-01, R01GM080469)
- Division of Graduate Education
- National Center for Research Resources (R01-RR16522, R01RR016522)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- phosphoproteomics, protein phosphorylation, multidimensional chromatography, iMac, hydrophilic interaction liquid chromatography, Saccharomyces cerevisiae, DNA damage response, Rad9, CD206, low-abundance proteins, post-translational modification
- MeSH terms
- Chromatography, DNA Damage, Models, Biological, Phosphopeptides, Phosphoproteins, Protein Binding, Saccharomyces cerevisiae, Cluster Analysis, Proteome, Proteomics, Metabolic Networks and Pathways
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