Zheng Naiyu, Taylor Kristin, Gu Huidong, Santockyte Rasa, Wang Xi-Tao, McCarty Jean, Adelakun Olufemi, Zhang Yan J, Pillutla Renuka, Zeng Jianing
Department of Translational Medicine, Bristol Myers Squibb, Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Anal Chem. 2020 Nov 3;92(21):14713-14722. doi: 10.1021/acs.analchem.0c03271. Epub 2020 Oct 13.
Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85-798.43 (amol/μg protein) for PD-L1 and 16.96-129.89 (amol/μg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.
尽管前景广阔，但通过液相色谱-串联质谱法（LC-MS/MS）对福尔马林固定石蜡包埋（FFPE）组织中的蛋白质生物标志物进行临床应用的生物分析仍然极具挑战性。在此，我们描述了一种灵敏且稳健的LC-MS/MS检测方法，用于定量FFPE肿瘤切片中的临床蛋白质生物标志物，该方法采用自动抗肽抗体免疫捕获，随后采用带有多同位素异构体反应监测（MIRM）技术的样品内校准曲线（ISCC）策略。带有稳定同位素标记（SIL）肽的MIRM的ISCC方法无需使用真实基质来绘制外部校准曲线，克服了基质效应，并在每个单独样品中验证了定量范围。具体而言，在脱石蜡、复水、抗原修复和匀浆后，向FFPE肿瘤组织中的蛋白质分析物中加入已知浓度的每种分析物的一种SIL肽，然后进行胰蛋白酶消化和抗肽免疫捕获富集，再进行基于MIRM-ISCC的LC-MS/MS分析。该方法已成功用于对来自肺癌、结直肠癌和头颈癌患者的15个代表性FFPE肿瘤样品中的程序性细胞死亡蛋白1（PD-1）和程序性细胞死亡配体1（PD-L1）进行灵敏定量。除一个样品外，使用该检测方法可对所有样品中的PD-L1和PD-1进行定量，PD-L1的浓度为27.85 - 798.43（amol/μg蛋白质），PD-1的浓度为16.96 - 129.89（amol/μg蛋白质）。这些结果总体上与免疫组织化学（IHC）数据一致，但也有一些例外。该方法证明了在FFPE组织中定量低丰度蛋白质生物标志物的可行性，具有更高的灵敏度、特异性和稳健性，并且作为IHC的一种正交分析方法在临床应用中显示出巨大潜力。
