Lysine and Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease

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.

带有翻译后修饰（post-translational modifications, PTMs）的蛋白质变体（Proteoforms），其功能多样性的充分挖掘，唯有通过精准同时定量多种翻译后修饰的分析策略方能实现。本研究提出一种基于数据非依赖采集质谱（data-independent acquisition-mass spectrometry, DIA-MS）的方法，可精准区分未修饰肽段与其携带翻译后修饰的对应肽段；该方法借助小型前体质量窗口，在二级质谱（MS2）采集阶段实现修饰肽变体之间的物理分离。本研究依托赖氨酸与精氨酸富集翻译后修饰的肽段检测文库及位点定位算法，可在单次质谱运行中同时完成7种翻译后修饰的定位与定量，涵盖单甲基化、二甲基化、三甲基化、乙酰化及琥珀酰化，同时实现总蛋白定量，且无需对实验样本进行富集处理。为评估该方法的生物学相关性，本研究将其应用于存在甲基化差异的非酒精性脂肪性肝炎（nonalcoholic steatohepatitis, NASH）小鼠模型的肝脏裂解物样本。本研究发现，甲基化与乙酰化的异常改变，连同总蛋白水平变化，共同催生了一项全新假说：翻译后修饰在非酒精性脂肪性肝炎的蛋白质合成与mRNA稳定性调控中发挥关键功能。