Protein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level. Pooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl- or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively. In the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation. This work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

中文翻译：

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患者结直肠癌组织中精氨酸甲基化的全蛋白质组鉴定。

蛋白质精氨酸甲基化反应由蛋白质精氨酸甲基转移酶 (PRMT) 催化，该修饰与包括癌症在内的各种疾病有关。目前，已经使用基于高分辨率质谱的蛋白质组学技术鉴定了数千个精氨酸甲基化位点。然而，尚未报道在蛋白质组水平上使用临床样品鉴定精氨酸甲基化。本研究的目的是在蛋白质组水平上鉴定结直肠癌 (CRC) 组织中的单甲基精氨酸 (MMA) 和不对称二甲基精氨酸 (ADMA) 位点。来自 10 名 II 期和 III 期患者的合并 CRC 组织样本被胰蛋白酶消化，这些消化物被进一步处理和冻干。使用单甲基或不对称二甲基精氨酸（分别为 MMA 或 ADMA）基序试剂盒，富含甲基精氨酸的肽，随后通过高分辨率 LC-MS/MS 进行分析。DLD1和HCT116结肠癌细胞用I型PRMTs抑制剂（MS023）单独或联合SN-38处理，通过水溶性四唑盐（WST-1）法测定药物对CRC细胞增殖和凋亡的影响和 FACS 分析，分别。在本研究中，从患者获得的 CRC 组织中鉴定了 272 种蛋白质的 455 个 MMA 位点和 155 种蛋白质的 314 个 ADMA 位点。此外，新鉴定了 PRMT 的 216 个甲基化位点和 75 个底物。这些结果揭示了 MMA 和 ADMA 位点在参与转录的核酸结合蛋白和蛋白复合物中的显着存在。探讨蛋白质精氨酸甲基化对结直肠癌增殖和凋亡的影响，MS023 用两种 CRC 细胞系处理。用不同浓度的 MS023 处理 48 小时后，CRC 细胞增殖被显着抑制，同时诱导细胞凋亡。此外，MS023 处理显着增强了 SN-38 对 CRC 细胞增殖的抑制作用。这项工作报告了对临床样本精氨酸甲基化的首次综合分析，并表明 I 型 PRMT 是 CRC 药物发现的潜在治疗靶点。