磷酸化蛋白體質譜法應用於微量組織;Microscale tissue profiling by mass spectrometry-based phosphoproteomics

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题名:	磷酸化蛋白體質譜法應用於微量組織;Microscale tissue profiling by mass spectrometry-based phosphoproteomics
作者:	張柏毅;Zhang, Bo-Yi
贡献者:	化學學系
关键词:	磷酸化蛋白體學;微量組織;非數據依賴擷取方法
日期:	2022-08-31
上传时间:	2022-10-04 11:01:13 (UTC+8)
出版者:	國立中央大學
摘要:	蛋白質磷酸化是一種重要的轉譯後修飾，負責調控訊息傳遞以及細胞生長等重要生物資訊，先進的質譜技術已經被證實是一種能夠深度分析磷酸化蛋白質體的方法，在細胞與生物樣本中達到31,000個磷酸化修飾位點。然而，目前對於臨床檢體的深度磷酸化蛋白質體分析仍然需要大量的組織(>30毫克)以及耗時的樣品製備步驟。具有高靈敏度且全面性的分析臨床樣本，例如福馬林石蠟包埋法(1毫克/部分)與活體穿刺(9到15毫克) 的策略仍然等待開發。因此，我們針對微量組織樣品發展與驗證一個高靈敏且流程精簡的磷酸化蛋白體分析方法，除此之外，我們更進一步將微量磷酸化蛋白體方法應用到人體乳癌組織檢體(0.5到2毫克)，其樣品量與福馬林石蠟包埋法所取得的樣品量相當，並且在癌症路徑中發現HER2和EGFR等重要的蛋白質。我們開發了一套利用Sodium laurate (SL)萃取蛋白質的方法，能夠省略耗時的蛋白質沉澱步驟並且同時保持高達百分之十的蛋白萃取效率。首先，我們使用人體肺癌細胞株PC9去驗證此方法的靈敏度及重複性，實驗結果顯示在1微克胜肽樣品中，我們能夠鑑定到超過7000條的磷酸化胜肽，並且保持高度的重複性(三重複的中位數CV<2 %)。由於在Stage-tip中組織樣品量的限制下，此方法只能應用到樣品量>0.5毫克的組織中。儘管樣品量僅有0.5毫克，我們仍然能鑑定到6300條的磷酸化胜肽，並且保有高達95%的磷酸化萃取專一性。為了進一步增加磷酸化蛋白體的分析深度，我們建立了一個老鼠肺臟圖譜的資料庫，包含DDA以及DIA原始圖譜)以進行Library-based的非數據依賴擷取方法(Data-Independent Acquisition，DIA)，此方法在0.5毫克的組織中能夠鑑定到23,272條磷酸化胜肽，相對於傳統的數據依賴擷取策略(Data-Dependent Acquisition，DDA)，在磷酸化胜肽鑑定上顯著提升3.7倍。最後，我們成功的將微量磷酸化蛋白體方法應用到人體臨床檢體上，研究癌症路徑中PI3K及PTEN等致癌基因，並且能夠在微量條件下以磷酸化修飾位點的訊息區分正常與癌症組織。總而言之，此微量組織策略能夠在24小時內完成(包含質譜儀器分析時間)，對於日常檢體分析具有龐大的優勢，未來這個方法將會應用到更多微量臨床組織與檢體，期許對於醫療診斷方面能夠提供更多的生物醫療的資訊。 ;Protein phosphorylation is one of the most important post-translational modifications (PTMs) regulating signal transduction in all aspects of life. Advanced mass spectrometry has been demonstrated as a promising tool for large-scale phosphoproteomics with the depth of 31,000 phosphosites in cells and clinical specimens. However, recent studies for deep phosphoproteomics in clinical specimens still required a large amount of tissues (>30 mg) and dozens of sample preparation steps. A strategy with high sensitivity capable of robust profiling for clinical specimens such as FFPE (1.0 mg/section) and needle biopsy (9.0-15 mg) awaits to be established. In this thesis, we developed a highly sensitive and streamlined approach for phosphoproteomics profiling of microscale tissues. We further applied this microscale phosphoproteomics method to human breast cancer tissues (0.5-2.0 mg) which the sample amount was equivalent to FFPE specimens and then revealed HER2 and EGFR important proteins in the cancer pathway. We have developed a streamlined approach using sodium laurate which could skip the time-consuming protein precipitation step and maintain high protein extraction efficiency. The PC9 cell line was used to evaluate the sensitivity and reproducibility. Identified 7,830 ± 57 phosphopeptides in only 1 μg peptide input and showed highly reproducibility in triplicates (median CV was less than 2 %). Due to the challenges of tissue sampling in the Stage-tip, application of this approach was limited to >0.5 mg (wet weight of tissue). Using 0.5 mg microscale mouse tissues, over 6,000 phosphopeptides were identified with high phosphopeptide enrichment specificity (95 %). To further increase the profiling coverage of tissue phosphoproteomics, we constructed a hybrid spectral library to incorporate library-based DIA strategy. Using 0.5 mg mouse samples, the strategy enhanced 3.7-fold coverage to reach 23,272 phosphopeptide identification than the conventional DDA method. Finally, we successfully applied this microscale approach to human breast cancer tissues to study the PI3K and PTEN oncogenes in cancer pathways that also separated normal and tumor samples with phosphosite distribution. Overall, the whole approach including LC-MS/MS analysis could be achieved in 24 hours, which provided a benefit for daily clinical research. The pipeline can be a general approach for phosphoproteomics profiling for microscale clinical tissues.
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