| 結束日期 |
2011-09-30 |
| 計畫單位 |
中醫學院中西醫結合研究所;中國附醫醫學研究部 |
| 計畫主持人 |
陳朝榮(Chao-Jung Chen) |
| 摘要 |
In this study, we developed a novel microwaveassisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) timeof- flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples. |
| 關鍵字 |
Carbon tape; Microwave-assisted protein preparation and digestion; Protein identification; Phosphopeptide; MALDI; Enrichment |
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