利用化學標定與質譜技術分析經由人類乳突病毒 E7 轉染癌細胞之第一型 MHC 胜肽

Abstract

第一型主要組織相容性複合體(major histocompatibility complex class I, MHC class I)呈現在所有的細胞表面，功能為提供來自外來病原或腫瘤相關的胜肽片段作為抗原，使免疫系統進行辨識。實驗使用同重元素相對和絕對定量(isobaric tags for relative and absolute quantitation, iTRAQ)的標定策略及質譜技術分析，經檸檬酸脫附後的人類乳突病毒(human papillomavirus, HPV) 轉染癌細胞之腫瘤相關抗原性胜肽。為了降低樣品複雜度並提高鑑定的動態範圍，使用奈米級液相層析質譜儀分析前會經由強陽離子交換層析(strong cationic exchange chromatography, SCX)、親水性交互作用層析(hydrophilic interaction chromatography, HILIC)及等電聚焦分級分離(solution isoelectric focusing, sIEF)對iTRAQ標定胜肽分離。數據分析上，首先以Swiss-Prot資料庫來確認，經質譜分析後的胜肽序列及其可信程度，再進一步使用MHC胜肽鍵結預測分析系統SYFPEITHI與immune epitope database (IEDB) 去預測胜肽與MHC class I分子間的鍵結親和性，最後以protein abundance across organisms (PaxDb) 與multi-omics profiling expression database (MOPED) 找尋MHC class I胜肽其來源蛋白質在各器官或疾病的表現量。癌細胞經由檸檬酸脫附後所得的樣品，以質譜鑑定並使用MHC胜肽鍵結預測分析系統，得到115段與MHC class I具有相關性的胜肽，並依據MHC的HLA*02:01型態篩選後得到78段胜肽，而其中FAG-、YVA-與YIP-此三段胜肽經由分析，確認會與MHC class I具有鍵結。本實驗提供ㄧ套以質譜分析為基礎的平台，去找尋MHC class I相關的胜肽，且T細胞若能對找尋到的MHC class I胜肽進行辨識並產生免疫反應，此胜肽對於腫瘤疫苗的發展將有極大的助益。

Major histocompatibility complex class I (MHC class I), which is present on the cell surface, play an important role in assisting immune system to recognize intracellular pathogens and tumor-derived peptide fragments. The goal of this study is to identify and to quantify tumor-associated peptides from HPV transformed cancer cell by citric acid treatment, isobaric tags for relative and absolute quantitation (iTRAQ) and mass spectrometric analysis. To reduce sample complexity for the quantitative dynamic range improvement, extracted MHC class I-bound peptides were fractionated by offline strong cationic exchange chromatography (SCX), hydrophilic interaction chromatography (HILIC) and solution isoelectric focusing (sIEF) before nanoLC mass spectrometric analysis. The tandem MS spectra were first searched against Swiss-Prot database for the possible MHC class I-associated peptide screening. Two algorithms, SYFPEITHI and immune epitope database (IEDB), were applied to calculate the binding affinity of MS-identified peptide sequence with MHC class I molecule. The results indicated that there were 115 MHC class I-associated peptides identified from the citric acid treated sample mixtures, and 78 of them were specific HLA*02:01-bound candidates. Among them, FAG-, YVA- and YIP- peptides were found to be stably bound with MHC class I by flow cytometry binding assay. Protein abundance across organisms (PaxDb) and multi-omics profiling expression database (MOPED) were also applied to validate the associate protein expression profiles of the predicted peptides in various organs and diseases. The proposed method provides an attractive alternative to discover native MHC class I-associated peptides by the MS-based platform. If these MHC class I-associated peptides can be recognized by T cells and be able to stimulate immune response, they will be of great assist in tumor vaccine development.