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Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis |
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| Authors: | Yoko Ino Eiji Kinoshita Emiko Kinoshita-Kikuta Tomoko Akiyama Yusuke Nakai Kohei Nishino Makoto Osada Akihide Ryo Hisashi Hirano Tohru Koike Yayoi Kimura |
| Affiliation: | 1. Advanced Medical Research Center, Yokohama City University, Yokohama, Japan Graduate School of Health Sciences, Gunma Paz University, Takasaki City, Gunma, Japan;2. Department of Human Nutrition, Faculty of Human Sciences, Hiroshima Bunkyo University, Hiroshima, Japan;3. Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan;4. Advanced Medical Research Center, Yokohama City University, Yokohama, Japan;5. Kuramoto Division, Technical Support Department, Tokushima University, Tokushima, Japan;6. Graduate School of Health Sciences, Gunma Paz University, Takasaki City, Gunma, Japan;7. Advanced Medical Research Center, Yokohama City University, Yokohama, Japan Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Japan |
| Abstract: | Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO2) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO2 and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO2 particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO2 particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis. |
| Keywords: | mass spectrometry phosphopeptide enrichment phos-tag proteomics titanium dioxide |