Hubbard Evan E, Shoff Thomas A, Hur Manhoi, Lambeth Tyler R, Chen Chengwei, Kung Ethan, Pan Bruce D, Lui Matthew K, Linares Javian R, Cantrell Lee S, Schey Kevin L, Julian Ryan R
Department of Chemistry, University of California, California, USA.
Department of Botany and Plant Sciences, University of California, Riverside, California, USA.
Aging Cell. 2025 Jun;24(6):e70028. doi: 10.1111/acel.70028. Epub 2025 May 1.
The eye lens is a unique tissue optimized for light transmission and refraction, necessitating dissolution of all organelles in mature fiber cells. This absence of organelles prevents protein turnover and leads to the accumulation of many spontaneous modifications over time. One modification that is oft overlooked is isomerization, despite its known impact on protein structure, interference with enzymatic activity, and association with disease. Prior analysis of isomerization in the lens has been limited to a small number of targets, consisting primarily of the highly abundant crystallin proteins. Proteomic coverage can be greatly increased by first depleting the crystallins and then employing state-of-the-art data-independent acquisition (DIA) mass spectrometry (MS). However, this approach has not been combined with data analysis methods capable of identifying isomers. By so doing, we identified hundreds of previously unreported, noncrystallin Asp isomer sites. To a lesser extent, isomerization was also detected at serine and glutamic acid, consistent with previous reports of relative isomerization propensities. Interestingly, we also identify histidine isomerization sites in a select number of peptides associated with metal adduction. We further analyzed our results according to primary sequence and secondary structure to explore factors potentially influencing isomerization. Finally, we found that while isomerization percents for individual proteins are modestly accurate predictor of age, inclusion of multiple isomerized sites affords a more accurate prediction of age, which may be useful for applications in forensics.
晶状体是一种独特的组织，经过优化以实现光的传输和折射，这使得成熟纤维细胞中的所有细胞器都溶解。细胞器的缺失阻止了蛋白质周转，并随着时间的推移导致许多自发修饰的积累。尽管异构化对蛋白质结构有已知影响、会干扰酶活性并与疾病相关，但它常常被忽视。此前对晶状体中异构化的分析仅限于少数靶点，主要由高度丰富的晶状体蛋白组成。通过首先去除晶状体蛋白，然后采用最先进的数据非依赖采集（DIA）质谱（MS），蛋白质组覆盖范围可以大大增加。然而，这种方法尚未与能够识别异构体的数据分析方法相结合。通过这样做，我们鉴定出了数百个以前未报道的非晶状体天冬氨酸异构化位点。在较小程度上，丝氨酸和谷氨酸也检测到了异构化，这与之前关于相对异构化倾向的报道一致。有趣的是，我们还在一些与金属加合相关的肽中鉴定出了组氨酸异构化位点。我们根据一级序列和二级结构进一步分析了我们的结果，以探索可能影响异构化的因素。最后，我们发现虽然单个蛋白质的异构化百分比对年龄的预测准确性一般，但纳入多个异构化位点能更准确地预测年龄，这可能对法医学应用有用。
