Accurate and efficient amino acid analysis for protein quantification using hydrophilic interaction chromatography coupled tandem mass spectrometry
Accurate and efficient amino acid analysis for protein quantification using hydrophilic interaction chromatography coupled tandem mass spectrometry
复制标题
DOI：
10.1186/s13007-019-0430-z
复制
发表时间：
2019-05-11
期刊：
影响因子：
5.1
通讯作者：
Allen, Doug K.
中科院分区：
文献类型：
--
作者：
Kambhampati, Shrikaar;Li, Jia;Allen, Doug K.
背景：用于从生物样品中定量蛋白质的方法通常是不准确的，需要注意尽量减少显著的可变性。错误是由于蛋白质制备和纯化过程中的损失以及干扰化合物或元素的错误检测造成的。氨基酸分析（AAA）涉及一系列色谱技术，可用于测量蛋白质水平，避免了一些困难，并提供特定的组成信息。然而，不稳定的衍生物，有毒且昂贵，反应不完全，色谱分离不充分，以及缺乏一种能充分回收所有氨基酸的单一水解方法，这些都阻碍了使用aaa进行精确的蛋白质定量。结果本研究采用基于亲水性相互作用色谱的方法分离了所有的蛋白质原氨基酸，包括异重化合物亮氨酸和异亮氨酸。用LC-MS/MS进行多反应监测。通过包含市售的同位素标记（C-13, N-15）氨基酸作为内部标准，我们采用了同位素稀释策略，用于基于氨基酸的蛋白质定量。用泛素、牛血清白蛋白（BSA）和大豆蛋白生物参比物质（SRM 3234； NIST）测试了三种水解方法，对三种蛋白质样品的蛋白质估计分别为86-103%、82-94%和90-99%。甲烷磺酸水解法提供了最佳的不稳定氨基酸回收率，包括半胱氨酸、蛋氨酸和色氨酸，这些不稳定氨基酸难以准确定量。结论异质生物样品中蛋白质含量和氨基酸组成的准确测定具有重要意义。色谱相和LC-MS/MS方法的最新进展，以及同位素标准的可用性，可以最大限度地减少分析困难，提高蛋白质定量。描述了一种可靠的高通量蛋白质定量和氨基酸组成分析方法。由于精确测量蛋白质的质量和数量是许多与作物改良有关的生物学研究的要求，或者更广泛地说，我们对生命系统代谢的理解，我们设想这种方法将具有广泛的适用性。
BackgroundMethods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, avoiding some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids hinder precise protein quantitation using AAA.ResultsIn this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC-MS/MS. Through inclusion of commercially available isotopically labeled (C-13, N-15) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86-103%, 82-94%, and 90-99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify.ConclusionsAccurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC-MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.