連續親和層析技術來分離純化磷酸化蛋白質胜肽

分析磷酸化蛋白質體的技術已發展至可以從複雜樣品中分離出磷酸化胜肽，像是IMAC(固定化金屬親和層析)、MOAC(金屬氧化物親和層析)、PA(奈米鑽石修飾聚精氨酸親和層析)等等，但是還是有部分的磷酸化胜肽不容易被偵測到，所以在偵測總體的磷酸化蛋白質體還是一項挑戰，我們發展出一個利用奈米鑽石上修飾聚精胺酸與二氧化鈦來做為純化出磷酸化胜肽的策略，且可以將單磷酸與多磷酸分開去做偵測，多磷酸的部分可以藉由奈米鑽石上修飾聚精胺酸來純化分離出來，而在同一個樣品中進一步用二氧化鈦可以濃縮出單磷酸胜肽，使用這個策略的結果所鑑定出的磷酸化胜肽數量多於使用單一個親和層析技術(IMAC,TiO2,PA)所鑑定出的數量還多，且此策略不需要加另外的生物藥劑而快速。

關鍵字

磷酸化胜肽親和層析；基質輔助雷射去脫附游離質譜；精胺酸

並列摘要

Despite recent technical advances, comprehensive characterization of protein phosphorylation remains a challenge. We explored the possibility using a two–step enrichment procedure for fractionation and selective identification of multiply and singly phosphorylated segments from enzymatic digestion of complex proteins. The procedure involved isolation of multiply phosphorylated peptides from solution using polyarginine-coated nonadiamond and subsequent isolation of singly phosphorylated peptides from the same pool of solution using TiO2-coated magnetic probes. The two types of probes can be independently characterized by MALDI mass spectrometry and provide complementary datasets for phosphoproteomic analysis. In addition, the total number of identified phosphopeptides obtained by the two-step enrichment procedure was almost doubled than that obtained by IMAC, TiO2, or PA affinity-based methods without the use of additional biological material. Improvements to comprehensive phosphopeptide identification via the two-step enrichment procedure are discussed. To our knowledge, this is the first demonstration of the two-step enrichment procedure for selective isolation and fractionation of multiply and singly phosphorylated peptides from the same pool of peptides solution.

並列關鍵字

phosphorylated peptides ； affinity chromatography ； MALDI-MS ； arginine

參考文獻

1.Wasinger, V.C.; Cordwell, S.J.; Cerpa-Poljak, A.; Yan, J.X., Gooley, A.A.; Wilkins, M.R.; Duncan, M.W.; Harris, R.; Williams, K.L.; Humphery-Smith, Electrophoresis 1995, 16, 1090.