陳頌方Sung Fang Chen段立平Li Ping Tuan2019-09-042018-8-192019-09-042013http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060042089S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/100392前列腺特異性抗原 (PSA) 是目前最成功且廣泛使用的前列腺癌 (PCa) 血清標誌物。但在重要診斷指標PSA濃度4~10 ng/mL的範圍內，它具有有限的能力在區分早期的前列腺癌與良性前列腺增生症 (BPH)，缺乏癌症特異性。近年來研究發現前列腺特異性抗原前體 (proPSA) 與前列腺癌更具相關性。ProPSA是血清游離PSA的特殊形式，由天然proPSA及其各種截斷的亞型組成，[-2] proPSA、[-5] proPSA及[-7] proPSA，而其各種截斷的亞型分子已被發現比天然proPSA與癌症有更好的相關性。(不是說不要分段嗎?)多反應監測質譜技術 (MRM-MS) 近年來頻繁地被應用於測量低含量的組織和生物體液中的生物標誌物，由於其高靈敏度和高特異性，實驗設計簡單和高度重複性。本研究建立一套利用免疫沉澱法搭配質譜多反應監測掃描技術之優化方法，檢測定量血清樣品中mature PSA、proPSA及truncated proPSA ([-5] proPSA)，藉此改善PSA對前列腺癌偵測之特異性。由於血清和血漿是目前最複雜的生物樣品，針對此，我們特別對免疫沉澱步驟當中所使用之實驗用管柱及緩衝溶液進行優化測試，以達到較高之純化效率，並且使用不同純化策略-免疫親和去除法¬比較純化目標蛋白質之效率。實驗結果證明此研究策略可有效純化血清中proPSA且定量檢測結果呈現良好之線性關係，proPSA及mature PSA的R2 >0.99，[-5] proPSA的R2 >0.99，偵測極限 (LOD) 和定量極限 (LOQ) 皆可達到proPSA濃度ng/mL的範圍內，對應血清中含量最高蛋白質的濃度，濃度低於6個數量級。此外，本研究策略在一次LC-MRM-MS實驗分析中可以檢測多個生物標誌物，包括成熟和前體形式的PSA。本研究開發此方法提供一個具吸引力的替代方案，藉由檢測定量血清樣品中proPSA及truncated proPSA以改善PSA對前列腺癌偵測之特異性。Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa). In the critical diagnostic range of 4~10 ng/mL it has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). More recently, promising data is emerging regarding one precursor form of free PSA, proPSA is associated with PCa. ProPSA is comprised of native proPSA as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, which have been shown to be more cancer-associated than native proPSA. Lately, multiple reaction monitoring mass spectrometry (MRM-MS) has been more frequently applied to measure low abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing. In this study, we developed and optimized a method for an immunoprecipitation-based platform and MRM-MS assay capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, we also optimized the vials and buffers used in the immunoprecipitation workflow to achieve such a level of sensitivity and compared the efficiency of protein purification with immunoaffinity depletion. The strategy we demonstrated the target proteins can be purified effectively; both limit of detection (LOD) and limit of quantitation (LOQ) of proPSA are able to reach nanogram/milliliter range, corresponding to a concentration that is 6-order lower than the concentration of the most abundant proteins in serum with good linearilty. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be performed in a single multiplexed analysis using LC-MRM-MS. We demonstrate that the strategy here provide an attractive alternative for reliably measuring proPSA to improve the detection of PCa.前列腺特異性抗原前列腺特異性抗原前體截斷前列腺特異性抗原前體免疫沉澱法多反應監測技術質譜前列腺癌PSAproPSAtruncated proPSAimmunoprecipitationmultiple reaction monitoringmass spectrometryPCa