評估胜肽分餾策略對人類肝癌腫瘤細胞PLC/PRF/5 之蛋白質體分析

Abstract

霰彈槍蛋白質體學(shotgun proteomics)是利用蛋白酶水解後的複雜胜肽樣品搭配串聯式質譜分析，即可準確鑑定複雜樣品中的蛋白質體，因此有效降低樣品複雜度的胜肽分餾方法在蛋白質體學的分析上是一門重要的技術。然而過去的研究中，許多文獻比較不同串聯式二維分析方法效率的文獻，本篇比較質譜分析前的四種胜肽分餾策略包括：強陽離子交換樹脂（SCX）、親水作用層析（HILIC）、鹼性逆相層析（alkaline-RP）、等電點聚焦分級分離儀（OFFGEL）和一控制組酸性逆相層析（acidic-RP），我們預期結合不同分離選擇性方法可達到良好的分離效率。根據分餾法不同的分離正交性(orthogonality)與液相層析效能，樣品去鹽後利用SCX x RP-LC分析得到最佳的蛋白質鑑定數目（佔總體蛋白質鑑定的96.54%），接下來是HILIC x RP-LC和alkaline-RP x RP-LC分析。值得注意的是去鹽後樣品利用SCX x RP-LC分析比樣品未經去鹽鑑定到更多的蛋白質（分別是1,990與1,375）。另外我們也發現結合所有胜肽分餾策略結果，鑑定到未重複胜肽（unique peptide）數目大幅增加，而鑑定的蛋白質數目並無顯著增加，因此對應蛋白質的未重複胜肽數目增加（胜肽數/蛋白質比例: 7.3），進而提升蛋白質身分鑑定的證據。不僅如此，鑑定到專一胜肽數目增加，對於後轉譯修飾的偵測例如: 磷酸化，與植基於胜肽之同位素標記定量方法例如: ICAT, iTRAQ, SILAC和TMT等，都有相當大的益處。此結果增加單一胜肽數對應蛋白質不僅提供良好的定量與定性資訊，也增加蛋白質序列的覆蓋率，可得到更高的可信度。

Shotgun approachhas been a commonlyapplied method for proteome analysis. In order to reduce the samplecomplexity prior to tandem mass spectrometry,peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few in which the relative separation efficiencies of 2D methodologies using complex biological samples are compared, a systematic evaluation was conducted in this study. Four different fractionation methods, including SCX（strongcation exchange）, HILIC（hydrophilic interaction liquid chromatography）, alkaline-RP and solution IEF prior to LC-MS/MS analysis wereinvestigated using PLC/PRF/5 cell lysate. Based on theircomplementary selectivities, it isexpected they canprovidea more comprehensive separation.SCX x RP-LC, using desalted samples, permitted the greatest number of proteins to be identifiedin this study（96.54% of the total proteins were identified）. The result was followed closely by HILIC x RP-LC and alkaline-RP x RP-LC. It is noteworthy that, when SCX x RP-LC was used after desalting the sample, significantly more proteins were able to be identified, compared with the non-desalted sample（1990 and 1375, respectively）. It isalso found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that were identified, compared with only a small increase in protein levels（average unique peptides/protein ratio is 7.3）. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving post-translational modifications and peptide-based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.