Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry |
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| Authors: | Sunkyu Choi Jaeyoon Kim Kun Cho Gunwook Park Jong Hyuk Yoon Sehoon Park Jong Shin Yoo Sung Ho Ryu Young Hwan Kim Jeongkwon Kim |
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| Affiliation: | 1. Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea;2. Department of Chemistry, Chungnam National University, Daejeon 305‐764, Republic of Korea;3. Mass Spectrometry Research Centre, Korea Basic Science Institute, Ochang, Cheong‐won 363‐883, Republic of Korea;4. Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305‐764, Republic of Korea |
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| Abstract: | Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe3O4) and titanium dioxide (TiO2) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe3O4 particles. In a subsequent step, any residual phosphopeptides were captured on TiO2 particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe3O4/TiO2 enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe3O4/TiO2 enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd. |
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