title: Compositional Analysis of Asymmetric and Symmetric Dimethylated H3R2 Using Liquid Chromatography-Tandem Mass Spectrometry-Based Targeted Proteomics.
abstract: Protein arginine methylation is one of the common post-translational modifications in cellular processes. To date, two isomeric forms of dimethylated arginine have been identified: asymmetric N(G),N(G)-dimethylarginine (aDMA), and symmetric N(G),N'(G)-dimethylarginine (sDMA). Evidence indicated that these isomers can coexist and have different or even opposite functions, with aDMA and sDMA forms of arginine 2 on histone H3 (i.e., H3R2me2a and H3R2me2s) being an example. Thus, specific detection and quantification of each isomeric form is important. Current methods are capable of predicting and detecting thousands of methylarginine sites in proteins, whereas differentiation and stoichiometric measurement of dimethylated protein isomers are still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has emerged as a promising technique for site-specific quantification of protein methylation using enzymatic peptides as surrogates of target proteins. However, it should be pointed out that a routine targeted proteomics strategy cannot easily distinguish sDMA- and aDMA-containing surrogate peptides due to their common nature. The estimated amount should be considered as the sum of both arginine dimethylated isomers. In this study, compositional analysis based on a linear algebra algorithm as an add-on to targeted proteomics was employed to quantify H3R2me2a and H3R2me2s (i.e., surrogate peptides of AR(me2a)TK(me1/2)QT and AR(me2s)TK(me1/2)QT). To achieve this simultaneous quantification, a targeted proteomics assay was developed and validated for each isomer first. With the slope and intercept of their calibration curves for each multiple reaction monitoring (MRM) transition, linear algebraic equations were derived. Using a series of mock mixtures consisting of isomers in varying concentrations, the reliability of the method was confirmed. Finally, the H3R2 dimethylation status was analyzed in normal MCF-10A cells, parental drug-sensitive MCF-7/WT cancer cells, and drug-resistant MCF-7/ADR cancer cells. Dimethylated H3R2 was also monitored in MCF-7/WT cells with the treatment of doxorubicin (DOX) for confirmation.
authors: School of Pharmacy, Nanjing Medical University , 818 Tian Yuan East Road, Nanjing, 211166, China.
authors: Qingqing
authors: Q
authors: Xu
authors: School of Pharmacy, Nanjing Medical University , 818 Tian Yuan East Road, Nanjing, 211166, China.
authors: Feifei
authors: F
authors: Xu
authors: School of Pharmacy, Nanjing Medical University , 818 Tian Yuan East Road, Nanjing, 211166, China.
authors: Liang
authors: L
authors: Liu
authors: School of Pharmacy, Nanjing Medical University , 818 Tian Yuan East Road, Nanjing, 211166, China.
authors: Yun
authors: Y
authors: Chen
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002853:Chromatography, Liquid; D004317:Doxorubicin / Q000737:chemistry / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D006657:Histones / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008745:Methylation; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D000903:Antibiotics, Antineoplastic; D006657:Histones; D004317:Doxorubicin