比較粒子填充式與聚合式分離管柱搭配奈流液相層析串聯質譜儀分析法於蛋白質體學研究

Abstract

在蛋白質體學的研究領域中，奈米級液相層析連接電噴灑游離源搭配串聯式質譜儀系統是相當重要的分析工具，當中以移動相不含鹽類的逆相層析法具有最好的儀器相容性與極佳的解析能力。本篇研究中，我們將採用shotgun proteomics分析策略，比較實驗室所自製的三種逆相層析毛細管分離管柱，並選用經胰蛋白酶水解之胜肽作為樣品，搭配串聯式質譜儀系統(QTOF)進行分析，藉此研究不同類型管柱對胜肽樣品分離特性。製備的毛細管分離管柱包括了兩種粒子填充式管柱totally porous silica C18 particle (3 μm, 100 Å)、HALO® fused core C18 particle (2.7 μm, 90 Å)，與一種聚合式管柱 SDVB monolithic (50 μm X 400 cm)；並於實驗中優化monolithic column製備之條件，包括聚合物種類、單體濃度、毛細管內徑、致孔劑組成、進樣量等條件逐一進行測試。實驗結果顯示40% SDVB單體濃度添加5% 1-propanol作為致孔劑配合內徑50 μm的毛細管所製備的管柱具有最佳的分離效能，且monolithic管柱可大幅度的降低系統運作壓力。搭配totally porous C18作為trapping column下，四公尺的SDVB column能夠有效降低co-elute干擾與減緩離子抑制現象，因此可大幅提升整體訊號強度，並增加質譜分析時所得到的譜圖數目；同時於GRAVY value分析中可發現SDVB與HALO® 能明顯提升親水性胜肽整體的鑑定比率； 同時亦透過萃取離子層析圖(XIC)來分析胎球蛋白(fetuin)的醣基化胜肽，結果顯示SDVB 與C18 相較於HALO®能鑑別較多的醣基化胜肽。這種新型的固定相分離介質具有廣泛的操作便利性與極佳的分離效能，相信能在蛋白質體學的研究中能夠展現出更完備的應用性。

Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) is a powerful tool in proteomic analysis. The preparation conditions for the SDVB-Monolithic column including the choice of monomer, inside diameter of capillary, and porogen are investigated. The performance of polymeric column was also compared and evaluated with micro particle-filled capillary columns, including a totally porous silica C18 column (75 μm x 100 mm, 3 μm, 100Å) and a HALO® fused core C18 column (75 μm x 100 mm, 2.7 μm, 90 Å). With all optimized conditions, the monolithic capillary column was prepared by in-situ polymerization of styrene and divinylbenzene (SDVB) inside a 4 meter-long, 50 μm i.d. fused silica capillary using 1-propanol as porogen. This continuous unitary porous structure provides more robust and high separation efficiency when comparing with the bead-based columns. Since the meter-long SDVB column could substantially reduce peptides co-elution and abate the ion suppression, the total ion current signal could be significantly enhanced with comparable flow rate and pressure. For the peptide identification in fetuin, the use of SDVB column in shotgun approach can identify more glycopeptides than HALO® column. The characterization of this novel monolithic mediawill be a promising addition to the stationary phase used in capillary column for proteome research.