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.

中文翻译：

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使用多同位素反应监测稳定同位素标记的分析物进行即时LC-MS / MS生物分析和定量蛋白质组学的样品内校准曲线。

一种新颖的样品内标定曲线（ISCC）方法，使用多种同位素同位素反应监测（MIRM）的稳定同位素标记（SIL）分析物的多个自然同位素同位素跃迁，用于即时液相色谱-串联质谱（LC-MS / MS）首次提出并证明了生物标志物，生物治疗剂和小分子化合物的生物分析。可以根据其子离子的同位素分布和中性损失来准确计算其MIRM通道中SIL分析物的理论同位素丰度。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精确测量。通过向每个研究样品中掺入已知量的SIL分析物，可以基于SIL分析物的选定MIRM通道中计算出的理论同位素丰度（分析物浓度当量）与每个单独研究样品中相应MIRM通道中测得的MS / MS峰面积之间的关系来建立ISCC。然后可以使用ISCC即时分别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。对MIRM–ISCC–LC-MS / MS方法进行了评估，并在此工作中进行了举例说明，并举例说明了通过免疫捕获和胰蛋白酶消化处理的人和猴血清中的蛋白质生物标记物；胰蛋白酶消化的人结肠组织匀浆中的三个替代肽；以及通过液-液萃取在人和大鼠血浆中提取的一种小分子药物。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，这种新颖的MIRM–ISCC–LC-MS / MS方法可以在许多潜在应用中提供准确而可靠的生物分析，尤其是在无法使用外部校准曲线的可靠矩阵的情况下。