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首页> 美国卫生研究院文献>Molecular Cellular Proteomics : MCP >ProteomeTools: Systematic Characterization of 21 Post-translational Protein Modifications by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Using Synthetic Peptides
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ProteomeTools: Systematic Characterization of 21 Post-translational Protein Modifications by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Using Synthetic Peptides

机译：ProteomeTools：使用合成肽通过液相色谱串联质谱（LC-MS / MS）对21种翻译后蛋白质修饰的系统表征

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The analysis of the post-translational modification (PTM) state of proteins using mass spectrometry-based bottom-up proteomic workflows has evolved into a powerful tool for the study of cellular regulatory events that are not directly encoded at the genome level. Besides frequently detected modifications such as phosphorylation, acetylation and ubiquitination, many low abundant or less frequently detected PTMs are known or postulated to serve important regulatory functions. To more broadly understand the LC-MS/MS characteristics of PTMs, we synthesized and analyzed ∼5,000 peptides representing 21 different naturally occurring modifications of lysine, arginine, proline and tyrosine side chains and their unmodified counterparts. The analysis identified changes in retention times, shifts of precursor charge states and differences in search engine scores between modifications. PTM-dependent changes in the fragmentation behavior were evaluated using eleven different fragmentation modes or collision energies. We also systematically investigated the formation of diagnostic ions or neutral losses for all PTMs, confirming 10 known and identifying 5 novel diagnostic ions for lysine modifications. To demonstrate the value of including diagnostic ions in database searching, we reprocessed a public data set of lysine crotonylation and showed that considering the diagnostic ions increases confidence in the identification of the modified peptides. To our knowledge, this constitutes the first broad and systematic analysis of the LC-MS/MS properties of common and rare PTMs using synthetic peptides, leading to direct applicable utility for bottom-up proteomic experiments.
机译：使用基于质谱的自下而上的蛋白质组学工作流对蛋白质的翻译后修饰（PTM）状态进行分析，已发展成为一种功能强大的工具，可用于研究不在基因组水平直接编码的细胞调节事件。除了经常检测到的修饰（例如磷酸化，乙酰化和泛素化）外，许多低丰度或检测频率较低的PTM已知或被假定具有重要的调节功能。为了更广泛地了解PTM的LC-MS / MS特性，我们合成并分析了约5,000个代表21种赖氨酸，精氨酸，脯氨酸和酪氨酸侧链天然修饰及其未修饰对应物的肽。分析确定了保留时间的变化，前体电荷状态的变化以及修饰之间搜索引擎得分的差异。使用11种不同的碎裂模式或碰撞能量评估PTM碎裂行为的变化。我们还系统地研究了所有PTM的诊断离子或中性丢失的形成，确认了10个已知的赖氨酸修饰并鉴定了5个新颖的诊断离子。为了证明在数据库搜索中包括诊断离子的价值，我们重新处理了赖氨酸巴豆酰化的公共数据集，并表明考虑诊断离子会增加对修饰肽段鉴定的信心。据我们所知，这构成了使用合成肽对常见和稀有PTM的LC-MS / MS特性进行的首次广泛而系统的分析，从而直接适用于自下而上的蛋白质组学实验。

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