Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.
Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.
复制标题
DOI：
10.1021/acs.analchem.2c03473
复制
发表时间：
2023-01-17
影响因子：
7.4
通讯作者：
Qu, Jun
中科院分区：
文献类型：
--
作者：
Pu, Jie;Xue, Chao;Huo, Shihan;Shen, Qingqing;Qu, Yang;Yang, Xinxin;An, Bo;Angel, Thomas E.;Chen, Zhuo;Mehl, John T.;Tang, Huaping;Yang, Eric;Sikorski, Timothy W.;Qu, Jun
基于液相色谱-质谱（LC-MS）的福尔马林固定石蜡包埋（FFPE）组织中目标蛋白的精确、绝对定量定量将极大地扩大药物/临床研究的样品可用性，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是必不可少的，但缺乏靶向、绝对定量的最佳程序；（ii）大多数FFPE样品是长期储存的；靶蛋白在储存过程中严重的免疫组化（IHC）信号丢失被广泛报道，而储存对基于lc - ms的方法的影响尚不清楚；（iii）制备校准/质量控制样品以确保FFPE组织中精确的靶向蛋白分析的适当策略仍然难以捉摸。利用FFPE组织中单克隆抗体（mAb）、抗原和40种组织标记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，可以在FFPE样品中进行准确和精确的靶向蛋白质定量。首先，我们演示了一种基于表面活性剂鸡尾酒的程序（f-SEPOD），从FFPE组织中提供高/可重复的目标特征肽回收率。其次，一项热加速降解研究在粗略估计的5年存储期内重现了蛋白质IHC信号的丢失，而所有靶标的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下降，但不太可能导致蛋白质的化学降解，这强烈表明FFPE组织的储存不会导致基于lc - ms的方法的显著定量偏差。第三，虽然传统的峰值提取校准方法会导致大量的负偏差，但使用ffpe处理的校准标准的新方法可以实现准确和精确的量化。通过该管道，我们首次进行了mAb及其靶点在FFPE组织中的药代动力学测量，其中FFPE与新鲜组织的时间过程显示出极好的相关性。
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 (f-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.
登录
查看更多内容
DOI：
10.1007/s10120-015-0566-0
发表时间：
2016-10
期刊：
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association
影响因子：
--
作者：
Catenacci DVT;Liao WL;Zhao L;Whitcomb E;Henderson L;O'Day E;Xu P;Thyparambil S;Krizman D;Bengali K;Uzzell J;Darfler M;Cecchi F;Blackler A;Bang YJ;Hart J;Xiao SY;Lee SM;Burrows J;Hembrough T
通讯作者：
Hembrough T
影响因子：
7.4
作者：
An, Bo;Zhang, Ming;Qu, Jun
通讯作者：
Qu, Jun
影响因子：
17.3
作者：
FRANKS, F
通讯作者：
FRANKS, F
影响因子：
4.4
作者：
Duan X;Young R;Straubinger RM;Page B;Cao J;Wang H;Yu H;Canty JM;Qu J
通讯作者：
Qu J
影响因子：
7.3
作者：
Coscia, Fabian;Doll, Sophia;Mann, Matthias
通讯作者：
Mann, Matthias