血清前處理大多以二維液相層析分離前處理，不同前處理方法會因樣品組成蛋白質特性差異而鑑定出不同部分的蛋白質。本篇研究是在二維胜肽液相層析分離前先行以陰陽離子交換與分子篩層析分離的多維處理手法，再接續著高解析質譜儀進行分析及資料庫比對。在蛋白質層級進行陰陽離子交換分離，收集陰離子蛋白質部分接續進行G-75分子篩管柱的液相層析分離，收集陰電性蛋白質的部分進行shotgun蛋白質分析。對蛋白質進行胰蛋白酶水解消化成胰蛋白酶胜肽，水解胜肽較複雜的部份則需再進行親水性管柱的分離收集，最後以C18逆相液相層析串聯質譜儀分析水解胜肽。資料庫搜尋軟體Mascot設定不同參數進行容錯搜尋及騙誘搜尋，對以不同方法處理的樣品進行統計比較。在這之中以多維分子篩管柱層析方式處理者擁有較多的鑑定結果，不論是在低含量蛋白質鑑定或是修飾位點的找尋皆有所優勢。
血清蛋白質中濃度在10~1000ppm的蛋白質有明顯鑑定數量上的提升，三種方法一共可以鑑定到1337種蛋白質。在處理大量資料時篩選胜肽分數排行第一且分數大於等於20分者，排除不確定的比對結果以提高胜肽的可信程度。對人血清蛋白的容錯搜尋可找到311個可能的修飾。針對G-75分子篩管柱所獲得數據分析Catechol estrogen修飾找到人血清蛋白上Lys519、Lys560兩個修飾點；找尋4-hydroxyl-2-nonenal修飾點則是可以找到11個修飾點。
多維分離血清樣品已是目前使用的前處理策略，但對於分子篩作為血清前處理手段之一的蛋白質體學分析方式尚未完善。本研究證明分子篩管柱對於複雜樣品剖析有顯著作用，唯結果看出仍有效果不足之處，未來持續改進即可應用於複雜樣品上分析低含量目標物。

Human serum protein analysis by liquid chromatography-mass spectrometry (LC-MS) is a difficult task due to highly complicated biological matrix and a broad dynamic range covering thousands of serum proteins. In this study, we developed a multiplex sample preparation and search strategy to improve proteomic identification of low abundant serum proteins and protein post-translational modifications by active metabolites. In this separation scheme, anionic serum proteins were fractionated by ion exchange followed by size exclusion chromatography. The collected protein fractions were digested by trypsin and analyzed by RPLC-LTQ-Orbitrap. The raw data were converted to a peak list which was used to search against human protein database based on fragment ion spectra (MS2) or error tolerance for non-targeted potential modifications. Preliminary results were filtered based on unique peptides, ≤ 1% false discovery rate (FDR), and a peptide probability score of 20. A total of 1337 proteins including low abundant serum proteins such as insulin and platelet factor 4 were identified. Furthermore, novel metabolite modifications of human serum albumin (HSA) by catechol estrogens (CEs) on Lys20, Lys519, Lys560, and His146 as well as by 4-hydroxyl-2-nonenal (4-HNE) on His338 were identified and confirmed by manual interpretation of the acquired spectra. These modifications may be used as potential biomarkers for the risk of oxidative stress or toxin exposure and our results provide useful data to develop targeting methods for their explorations.

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