Medizinisches Proteom-Center (MPC), Ruhr-Universität Bochum, Bochum, Germany.
Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum, Bochum, Germany.
Methods Mol Biol. 2021;2228:145-157. doi: 10.1007/978-1-0716-1024-4_11.
Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).
靶向蛋白质组学是一种高效的方法，可以高灵敏度和准确性地定量感兴趣的蛋白质。靶向方法最初是为三重四极杆仪器建立的，但允许对 MS/MS 碎片离子进行高分辨率和精确质量测量的混合仪器的出现，使得平行反应监测 (PRM) 的发展成为可能。在 PRM 分析中，特定的肽被用作复杂样品中蛋白质的代表进行测量，同时获取完整的产物离子谱，从而可以对肽进行鉴定和定量。理想情况下，将相应的稳定同位素标记的肽掺入到分析的样品中，以解释技术变化并提高精度。在这里，我们描述了 PRM 测定法的开发，包括选择合适的肽，这些肽满足作为靶向蛋白质的独特替代物的标准。我们描述了方法开发的顺序步骤和校准曲线的生成。此外，我们还介绍了用于确定线性浓度范围和定量下限 (LOQ) 的开放访问工具 CalibraCurve。
