Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, United States.
Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, New York 14214, United States.
Anal Chem. 2023 Jan 17;95(2):924-934. doi: 10.1021/acs.analchem.2c03473. Epub 2022 Dec 19.
Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.
准确、绝对的液相色谱-质谱（LC-MS）定量分析福尔马林固定石蜡包埋（FFPE）组织中的目标蛋白将极大地扩展药物/临床研究的样本可用性，但由于以下问题仍然具有挑战性：（i）从 FFPE 组织中高效/定量回收目标特征肽对于靶向、绝对定量至关重要，但缺乏最佳的程序；（ii）大多数 FFPE 样本是长期储存的；目标蛋白在储存过程中的免疫组织化学（IHC）信号丢失广泛报道，而储存对基于 LC-MS 的方法的影响尚不清楚；（iii）确保在 FFPE 组织中准确进行靶向蛋白分析的校准/质量控制样本的适当制备策略仍然难以捉摸。使用 FFPE 组织中的单克隆抗体（mAb）、抗原和 40 种组织标志物的靶向定量作为模型系统，我们广泛研究了这些问题，并开发了一种基于 LC-MS 的策略，能够实现 FFPE 样本中准确和精确的靶向蛋白定量。首先，我们证明了基于表面活性剂鸡尾酒的程序（-SEPOD）可提供高/可重复的目标特征肽从 FFPE 组织中的回收。其次，在大约估计的 5 年储存期内进行的热加速降解研究重现了蛋白 IHC 信号的丢失，而所有目标的 LC-MS 信号保持不变。这表明 FFPE 组织的储存主要导致免疫反应性降低，但不太可能导致蛋白质化学降解，这强烈表明 FFPE 组织的储存不会对基于 LC-MS 的方法造成显著的定量偏差。第三，虽然传统的加标提取方法会导致显著的负偏差，但使用 FFPE 处理的校准标准的新方法可实现准确和精确的定量。通过该流程，我们首次在 FFPE 组织中进行了 mAb 及其靶标的药代动力学测量，FFPE 与新鲜组织的时间曲线显示出极好的相关性。
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