CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090, Vienna, Austria.
Department of Laboratory Medicine, Medical University of Vienna , 1090, Vienna, Austria.
Anal Chem. 2017 Apr 4;89(7):3847-3852. doi: 10.1021/acs.analchem.6b05065. Epub 2017 Mar 15.
Mass spectrometric-based proteomics is a powerful tool to analyze post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of post-translational modifications that are poorly characterized with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehyde-modified proteins and peptides. Malondialdehyde is among the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders, and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimized for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimized procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localize the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.
基于质谱的蛋白质组学是分析翻译后修饰蛋白质的有力工具。脂质过氧化产物引起的羰基化修饰是一类翻译后修饰，其蛋白质靶标和功能特征研究得很差。这在一定程度上是由于缺乏专门的基于质谱的技术来促进这些修饰的分析。在这里，我们提出了一种全面的方法来鉴定丙二醛修饰的蛋白质和肽。丙二醛是脂质过氧化产物中最丰富的一种；并且蛋白质上的丙二醛衍生加合物与心血管疾病、神经退行性疾病和其他临床病症有关。我们的综合方法针对整体蛋白质组学工作流程的三个层次：（i）通过采用靶向富集策略进行样品制备；（ii）通过使用优化的梯度进行高效液相色谱分离修饰肽；和（iii）通过串联质谱提高非常低丰度肽的光谱质量。通过将优化后的程序应用于用低量丙二醛修饰的蛋白质进行全细胞裂解物，我们能够鉴定多达 350 种不同的修饰肽，并将修饰定位到特定的赖氨酸残基上。该方法允许全面分析丙二醛修饰的蛋白质。
