Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089, United States.
Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90089, United States.
J Proteome Res. 2020 Aug 7;19(8):3123-3129. doi: 10.1021/acs.jproteome.0c00116. Epub 2020 Jun 1.
Protein arginine methylation regulates diverse biological processes including signaling, metabolism, splicing, and transcription. Despite its important biological roles, arginine dimethylation remains an understudied post-translational modification. Partly, this is because the two forms of arginine dimethylation, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are isobaric and therefore indistinguishable by traditional mass spectrometry techniques. Thus, there exists a need for methods that can differentiate these two modifications. Recently, it has been shown that the ADMA and SDMA can be distinguished by the characteristic neutral loss (NL) of dimethylamine and methylamine, respectively. However, the utility of this method is limited because the vast majority of dimethylarginine peptides do not generate measurable NL ions. Here, we report that increasing the normalized collision energy (NCE) in a higher-energy collisional dissociation cell increases the generation of the characteristic NLs that distinguish ADMA and SDMA. By analyzing both synthetic and endogenous methyl-peptides, we identify an optimal NCE value that maximizes NL generation and simultaneously improves methyl-peptide identification. Using two orthogonal methyl-peptide enrichment strategies, high pH strong cation-exchange and immunoaffinity purification, we demonstrate that the optimal NCE improves NL-based ADMA and SDMA annotation and dimethyl-peptide identifications by 125% and 17%, respectively, compared to the standard NCE. This simple parameter change will greatly facilitate the identification and annotation of ADMA and SDMA in mass spectrometry-based methyl-proteomics to improve our understanding of how these modifications differentially regulate protein function. All raw data have been deposited in the PRIDE database with accession number PXD017193.
精氨酸的蛋白质甲基化调控多种生物学过程，包括信号转导、代谢、剪接和转录。尽管其具有重要的生物学作用，但精氨酸二甲基化仍然是一种研究不足的翻译后修饰。部分原因是两种形式的精氨酸二甲基化，不对称二甲基精氨酸（ADMA）和对称二甲基精氨酸（SDMA），是等质量的，因此无法通过传统的质谱技术区分。因此，需要开发能够区分这两种修饰的方法。最近，已经表明 ADMA 和 SDMA 可以通过二甲胺和甲胺的特征中性丢失（NL）分别区分。然而，该方法的实用性有限，因为绝大多数二甲基精氨酸肽不会产生可测量的 NL 离子。在这里，我们报告说，在更高能量碰撞解离池中增加归一化碰撞能量（NCE）可以增加区分 ADMA 和 SDMA 的特征 NL 的产生。通过分析合成和内源性甲基肽，我们确定了一个最佳的 NCE 值，该值最大限度地提高了 NL 的产生，同时提高了甲基肽的鉴定。使用两种正交的甲基肽富集策略，高 pH 强阳离子交换和免疫亲和纯化，我们证明与标准 NCE 相比，最佳 NCE 可将基于 NL 的 ADMA 和 SDMA 注释以及二甲基肽鉴定分别提高 125%和 17%。这种简单的参数变化将极大地促进基于质谱的甲基蛋白质组学中 ADMA 和 SDMA 的鉴定和注释，从而提高我们对这些修饰如何差异调节蛋白质功能的理解。所有原始数据均已存入 PRIDE 数据库，登录号为 PXD017193。
