Advanced Clinical Biosystems Research Institute, The Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, California 90048, United States.
Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.
J Proteome Res. 2020 Oct 2;19(10):4163-4178. doi: 10.1021/acs.jproteome.0c00685. Epub 2020 Sep 23.
Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.
含翻译后修饰（PTM）的蛋白异构体代表了一种功能多样性，只有通过对多种 PTM 进行精确的同时定量分析，才能充分发挥其功能。在这里，我们提出了一种通过数据非依赖性采集-质谱法从其含 PTM 的对应物中准确区分未修饰肽的方法，利用小的前体质量窗口，在 MS2 采集过程中物理分离修饰肽。我们利用富含赖氨酸和精氨酸 PTM 的肽检测文库和位点定位算法，在单次 MS 运行中同时定位和定量七种 PTM，包括单、二和三甲基化、乙酰化和琥珀酰化，以及总蛋白质定量，而无需富集实验样本。为了评估生物学相关性，该方法应用于不同甲基化非酒精性脂肪性肝炎（NASH）小鼠模型的肝裂解物。我们报告说，甲基化和乙酰化的改变以及总蛋白的变化共同提出了一个新的假设，即 PTM 在 NASH 中的蛋白质合成和 mRNA 稳定性中具有调节功能。
