Reddy Raven J, Curran Timothy G, Zhang Yi, White Forest M
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Methods Mol Biol. 2016;1410:281-92. doi: 10.1007/978-1-4939-3524-6_17.
Mass spectrometry, when coupled to on-line separation such as liquid chromatography or capillary electrophoresis, enables the identification and quantification of protein expression and post-translational modification changes under diverse conditions. To date most of the methods for mass spectrometry-based quantification have either provided relative quantification information (e.g., comparison to a selected condition) or utilized one-point calibration curves, or calibration curves in a different biological matrix. Although these quantitative methods have been used to generate insight into the differences between biological samples, additional biological insight could be gained by accurately measuring the absolute quantity of selected proteins and protein modifications. To address this challenge, we have developed the MARQUIS (Multiplex Absolute Regressed Quantification with Internal Standards) method, designed to provide absolute quantification for potentially hundreds of peptides across multiple samples in a single analysis, using a multi-point internal calibration curve derived from synthetic, isotopically distinct standard peptides.
质谱联用在线分离技术，如液相色谱或毛细管电泳，能够在不同条件下对蛋白质表达及翻译后修饰变化进行鉴定和定量。迄今为止，大多数基于质谱的定量方法要么提供相对定量信息（例如，与选定条件进行比较），要么使用单点校准曲线，或者在不同生物基质中的校准曲线。尽管这些定量方法已用于深入了解生物样品之间的差异，但通过准确测量选定蛋白质和蛋白质修饰的绝对量，还可以获得更多生物学见解。为应对这一挑战，我们开发了MARQUIS（带内标的多重绝对回归定量）方法，该方法旨在通过使用由合成的、同位素不同的标准肽得出的多点内标校准曲线，在单次分析中对多个样品中的数百种肽进行绝对定量。
