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首页> 外文期刊>Analytical Biochemistry: An International Journal of Analytical and Preparative Methods >Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance
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Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance

机译：使用稳定同位素和质谱对蛋白质翻译后修饰进行定量。二。性能

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摘要

In this report, we examine the performance of a mass spectrometry (MS)-based method for quantification of protein posttranslational modifications (PTMs) using stable isotope labeled internal standards. Uniform labeling of proteins and highly similar behavior of the labeled vs nonlabeled analyte pairs during chromatographic separation and electrospray ionization (ESI) provide the means to directly quantify a wide range of PTMs. In the companion report (Jiang et al., Anal. Biochem., 421 (2012) 506-516.), we provided principles and example applications of the method. Here we show satisfactory accuracy and precision for quantifying protein modifications by using the SILIS method when the analyses were performed on different types of mass spectrometers, such as ion-trap, time-of-flight (TOF), and quadrupole instruments. Additionally, the stable isotope labeled internal standard (SILIS) method demonstrated an extended linear range of quantification expressed in accurate quantification up to at least a 4 log concentration range on three different types of mass spectrometers. We also demonstrate that lengthy chromatographic separation is no longer required to obtain quality results, offering an opportunity to significantly shorten the method run time. The results indicate the potential of this methodology for rapid and large-scale assessment of multiple quality attributes of a therapeutic protein in a single analysis.
机译：在本报告中，我们使用稳定同位素标记的内标检查了基于质谱（MS）定量蛋白质翻译后修饰（PTM）的方法的性能。蛋白质的均匀标记以及色谱分离和电喷雾电离（ESI）期间标记的和未标记的分析物对的高度相似行为，提供了直接量化各种PTM的方法。在伴随报告中（Jiang等人，Anal。Biochem。，421（2012）506-516。），我们提供了该方法的原理和示例应用。当在不同类型的质谱仪（例如离子阱，飞行时间（TOF）和四极杆仪器）上进行分析时，我们在使用SILIS方法定量蛋白质修饰时显示出令人满意的准确性和精密度。此外，稳定同位素标记内标（SILIS）方法证明了在三种不同类型的质谱仪上以精确定量表示的扩展线性定量范围，直至至少4 log浓度范围。我们还证明不再需要冗长的色谱分离来获得高质量的结果，这为大大缩短方法的运行时间提供了机会。结果表明该方法有可能在单个分析中快速大规模评估治疗性蛋白质的多个质量属性。

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