Duriez Elodie, Trevisiol Stephane, Domon Bruno
Luxembourg Clinical Proteomics Center, CRP-Santé , 1 A-B rue Thomas Edison, Strassen 1445, Luxembourg.
J Proteome Res. 2015 Feb 6;14(2):728-37. doi: 10.1021/pr500764e. Epub 2014 Dec 3.
Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.
基于同位素稀释和质谱分析的肽和蛋白质定量在生物标志物测量及系统生物学应用中被广泛采用。此类定量分析的准确性和可靠性取决于稳定同位素标记标准品的质量。尽管使用稳定同位素标记肽进行定量很精确，但结果的准确性可能会因内标物的纯度、稳定性和配方以及其浓度的测定而受到严重偏差。在此，我们描述了一种在单次液相色谱 - 质谱分析中重新校准稳定同位素标记肽的快速且经济高效的方法。该方法基于在串联多肽标准品的胰蛋白酶消化过程中，蛋白质参考肽（用作目标蛋白质的替代物）和通用报告肽的等摩尔释放。通过对尿液样本中两种临床重要蛋白质的定量突出了使用此类串联多肽标准品生成的数据的质量和准确性，并与使用传统稳定同位素标记参考肽获得的结果进行了比较。此外，还描述了通用报告肽标准品在复杂样品中的应用。
