LeBlanc André, Michaud Sarah A, Percy Andrew J, Hardie Darryl B, Yang Juncong, Sinclair Nicholas J, Proudfoot Jillaine I, Pistawka Adam, Smith Derek S, Borchers Christoph H
University of Victoria - Genome BC Proteomics Centre , Victoria, BC V8Z 7X8, Canada.
Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, McGill University , Montreal, Quebec H3A 0G4, Canada.
J Proteome Res. 2017 Jul 7;16(7):2527-2536. doi: 10.1021/acs.jproteome.7b00094. Epub 2017 Jun 2.
When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.
在基础研究、生物医学研究以及临床应用中对内源性血浆蛋白进行定量时，需要精确、可重复且稳健的检测方法。当与稳定同位素标记肽结合使用时，自下而上策略中对肽段的靶向检测是最常见且精确的基于质谱的定量方法。然而，在测量血浆中的蛋白质时，未知的内源性水平阻碍了最佳校准策略的实施，因为没有空白基质可用。因此，不同实验室采用了几种替代校准策略。在本研究中，将这些方法与一种新方法进行了比较，该新方法针对每个要定量的内源性肽使用两种不同的稳定同位素标记标准（SIS）肽同位素异构体，从而能够在混合人血浆中制备外部校准曲线以及质量控制样品，而不受内源性肽的干扰。这种策略提高了检测的分析性能，并能够监测检测的准确性，这也有助于方法的开发和验证。
