藉由蛋白質體學中運用的奈流液相層析質譜技術探討全多孔式、熔融核心式與聚合物單層式管柱之分離特性
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2015
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奈流液相層析連接電噴灑游離搭配串聯式質譜系統已是現今蛋白質體學主流的分析工具之一。在液相層析部分，逆相層析法與電噴灑質譜分析具有高度的相容性，且提供良好的解析力，使之成為了最常搭配質譜分析使用的分離策略。本篇實驗的研究目標，在於探討三種不同逆相液相層析管柱對於蛋白質水解產物胜肽的分離特性。三種管柱分別是填充粒子式的totally porous C18 column與HALO®2.7 Fused-core® C18 column，以及苯乙烯與乙二烯苯的共聚物製成之單層式SDVB column。選用的分析物分別來自牛血清白蛋白、胎球蛋白與酪蛋白經酵素水解之胜肽產物，藉由液相層析分離，電噴灑游離法離子化後進入質譜分析，由質譜的數據觀測各分析物在液相層析管柱分離下之特性。使用三種管柱搭配質譜儀分析一般胜肽樣品所鑑定到的胜肽數量無顯著差異，僅HALO Fused-core管柱之分離效率略優於其他管柱。鑑定胰蛋白酶水解之胎球蛋白中的醣基化胜肽時，使用聚合成之SDVB管柱擁有最佳的分離能力，以及鑑定到20種不同的醣基化胜肽；傳統的totally porous C18鑑定到19種醣基化胜肽，但無法有效分離具相同胜肽骨幹的醣基化胜肽。可惜的是此三種管柱對於高度親水性的樣品滯留能力不佳，皆無法順利分離氮-醣鏈醣類樣品。使用單層式SDVB管柱對於水解酪蛋白樣品中磷酸化胜肽鑑定效果最好，訊號強度約為其餘兩種管柱的二倍，亦具備有效分離不同數量磷酸化修飾位於同一段胜肽的能力。實驗結果導向此種聚合成單層式SDVB管柱對於親水性的後轉譯修飾的胜肽擁有最佳的分離效率。我們將更進一步的探討此種新型的聚合材質之特性與效能，它有潛力在蛋白質體學研究中成為另一種具有吸引力的毛細管柱之固定相材質。
Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry is the major platform for proteomic analysis. Reverse phase chromatography also provides high resolving power and good compatibility with electrospray mass spectrometry. It becomes a widely applied online fractionation strategy for proteome research. In this study, the characteristics of three stationary phases used in reverse phase columns (totally porous C18, HALO®2.7 Fused-core® C18 and monolithic SDVB) were investigated for peptides, glycopeptides, phosphopeptides and glycans separation. Structures of totally porous C18 and HALO®2.7 Fused-core® C18 columns are bead-based and monolithic SDVB column are styrene -divinylbenzene copolymer. For regular peptide identification, performances of these three columns were insignificant. For glycopeptides, monolithic SDVB column gave the highest separation efficiency and a total of 20 N-linked glycopeptides were identified in tryptic fetuin. SDVB column is capable of separating the peptides with same backbone but different glycosylation motifs. Unfortunately, all three columns were unable to separate N-linked glycans form fetuin. In addition, SDVB column enhance more than 2-fold intensity for phosphopeptide detection. Besides, it can also separate phosphopeptides with various phosphorylation sites. The results demonstrated in this study show that SDVB column possesses great potential for hydrophilic peptide separation. The characterization and performance of this novel monolithic media will be further examined and it may be a promising addition to the stationary phase used in capillary column for proteome research.
Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry is the major platform for proteomic analysis. Reverse phase chromatography also provides high resolving power and good compatibility with electrospray mass spectrometry. It becomes a widely applied online fractionation strategy for proteome research. In this study, the characteristics of three stationary phases used in reverse phase columns (totally porous C18, HALO®2.7 Fused-core® C18 and monolithic SDVB) were investigated for peptides, glycopeptides, phosphopeptides and glycans separation. Structures of totally porous C18 and HALO®2.7 Fused-core® C18 columns are bead-based and monolithic SDVB column are styrene -divinylbenzene copolymer. For regular peptide identification, performances of these three columns were insignificant. For glycopeptides, monolithic SDVB column gave the highest separation efficiency and a total of 20 N-linked glycopeptides were identified in tryptic fetuin. SDVB column is capable of separating the peptides with same backbone but different glycosylation motifs. Unfortunately, all three columns were unable to separate N-linked glycans form fetuin. In addition, SDVB column enhance more than 2-fold intensity for phosphopeptide detection. Besides, it can also separate phosphopeptides with various phosphorylation sites. The results demonstrated in this study show that SDVB column possesses great potential for hydrophilic peptide separation. The characterization and performance of this novel monolithic media will be further examined and it may be a promising addition to the stationary phase used in capillary column for proteome research.
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液相層析連結質譜儀, totally porous C18, Fused-core, 單層式管柱, 苯乙烯與乙二烯苯共聚物, LC-MS/MS, totally porous, Fused-core, monolithic, SDVB