Bioanalytical Sciences, Research & Development , Bristol-Myers Squibb , Route 206 & Province Line Road , Princeton , New Jersey 08543 , United States.
Anal Chem. 2019 Feb 5;91(3):2536-2543. doi: 10.1021/acs.analchem.8b05656. Epub 2019 Jan 22.
A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM-ISCC-LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid-liquid extraction. The potential applications of the MIRM-ISCC-LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM-ISCC-LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.
提出并首次展示了一种使用稳定同位素标记（SIL）分析物的多个天然存在的同位素标记跃迁的多同位素反应监测（MIRM）进行样品内校准曲线（ISCC）的新方法，用于即时液相色谱-串联质谱（LC-MS/MS）生物分析生物标志物、生物治疗剂和小分子化合物。可以根据其子离子和中性丢失的同位素分布准确计算 SIL 分析物在其 MIRM 通道中的理论同位素丰度。这些 MIRM 通道中的同位素丰度也可以使用三重四极杆质谱仪准确测量。通过向每个研究样品中加入已知量的 SIL 分析物，可以根据 SIL 分析物的选定 MIRM 通道中的计算理论同位素丰度（分析物浓度当量）与每个研究样品中相应 MIRM 通道中的测量 MS/MS 峰面积之间的关系建立 ISCC。然后可以使用 ISCC 立即单独计算每个研究样品的分析物浓度，而无需使用外部校准曲线。本文通过免疫捕获和胰蛋白酶消化处理的人血清和猴血清中蛋白质生物标志物的定量、胰蛋白酶消化的人结肠组织匀浆中的三个替代肽以及液液萃取的人血浆和大鼠血浆中的小分子药物的定量示例，评估并展示了 MIRM-ISCC-LC-MS/MS 方法。本文还讨论了 MIRM-ISCC-LC-MS/MS 方法在定量蛋白质组学、临床实验室和其他领域的潜在应用。无需使用外部校准曲线，这种新的 MIRM-ISCC-LC-MS/MS 方法可以在许多潜在应用中提供准确可靠的生物分析，特别是在没有外部校准曲线真实基质的情况下。
