The Barnett Institute of Chemical and Biological Analysis of Northeastern University, Boston, Massachusetts, United States, Massachusetts General Hospital Cancer Center, Boston, Massachusetts, United States, and Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, United States.
Anal Chem. 2010 Nov 1;82(21):8998-9005. doi: 10.1021/ac101981t. Epub 2010 Oct 11.
Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and interassay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers.
液相色谱-选择反应监测（LC-SRM）是一种高度特异性和灵敏的质谱（MS）技术，广泛应用于选择性鉴定和验证复杂生物样本中的候选标志物。然而，为了使 LC-SRM 方法具有这些属性，需要对样品处理进行特定于目标的优化，以在 LC-SRM 之前降低分析物的复杂性。在这项研究中，我们开发了一个靶向平台，包括磁性珠上的蛋白质免疫亲和富集和 LC-SRM，用于测量肾细胞癌（RCC）细胞系（PRC3）中的碳酸酐酶 12（CA12）蛋白，这是一种 RCC 的候选生物标志物，其蛋白水平的表达以前没有报道过。针对作为 CA12 蛋白替代标志物选择的特征肽进行了样品处理和 LC-SRM 分析的优化。我们使用 LC-SRM 与稳定同位素稀释相结合，在低飞摩尔范围内实现了定量下限，足以测量具有良好的内和间测定准确性和精密度（≤17%）的临床相关生物标志物。我们的结果表明，使用定量免疫亲和捕获方法可以提供特异性、准确性和稳健的检测方法，适用于高通量验证潜在的生物标志物。
