Wanigasekara Maheshika S K, Huang Xiaojun, Chakrabarty Jayanta K, Bugarin Alejandro, Chowdhury Saiful M
Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019-0065, United States.
ACS Omega. 2018 Oct 26;3(10):14229-14235. doi: 10.1021/acsomega.8b01729. eCollection 2018 Oct 31.
Modification of arginine residues using dicarbonyl compounds is a common method to identify functional or reactive arginine residues in proteins. Arginine undergoes several kinds of posttranslational modifications in these functional residues. Identifying these reactive residues confidently in a protein or large-scale samples is a very challenging task. Several dicarbonyl compounds have been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione. However, tracking these reactive arginine residues in a protein or large-scale protein samples using a chemical labeling approach is very challenging. Thus, the enrichment of modified peptides will provide reduced sample complexity and confident mass-spectrometric data analysis. To pinpoint arginine-labeled peptide efficiently, we developed a novel arginine-selective enrichment reagent. For the first time, we conjugated an azide tag in a widely used dicarbonyl compound cyclohexanedione. This provided us the ability to enrich modified peptides using a bio-orthogonal click chemistry and the biotin-avidin affinity chromatography. We evaluated the reagent in several standard peptides and proteins. Three standard peptides, bradykinin, substance P, and neurotensin, were labeled with this cyclohexanedione-azide reagent. Click labeling of modified peptides was tested by spiking the peptides in a myoglobin protein digest. A protein, RNase A, was also labeled with the reagent, and after click chemistry and biotin-avidin affinity chromatography, we identified two selective arginine residues. We believe this strategy will be an efficient way for identifying functional and reactive arginine residues in a protein or protein mixtures.
使用二羰基化合物修饰精氨酸残基是鉴定蛋白质中功能性或反应性精氨酸残基的常用方法。在这些功能性残基中，精氨酸会经历几种翻译后修饰。在蛋白质或大规模样本中准确鉴定这些反应性残基是一项极具挑战性的任务。已经使用了几种二羰基化合物，其中最有效的是苯乙二醛和环己二酮。然而，使用化学标记方法在蛋白质或大规模蛋白质样本中追踪这些反应性精氨酸残基非常具有挑战性。因此，富集修饰后的肽段将降低样品复杂性并实现可靠的质谱数据分析。为了有效地确定精氨酸标记的肽段，我们开发了一种新型的精氨酸选择性富集试剂。我们首次在广泛使用的二羰基化合物环己二酮中连接了一个叠氮化物标签。这使我们能够使用生物正交点击化学和生物素-抗生物素蛋白亲和色谱法富集修饰后的肽段。我们在几种标准肽和蛋白质中评估了该试剂。三种标准肽，缓激肽、P物质和神经降压素，用这种环己二酮-叠氮化物试剂进行了标记。通过将肽段添加到肌红蛋白蛋白消化物中来测试修饰后肽段的点击标记。一种蛋白质，核糖核酸酶A，也用该试剂进行了标记，经过点击化学和生物素-抗生物素蛋白亲和色谱法后，我们鉴定出了两个选择性精氨酸残基。我们相信这种策略将是鉴定蛋白质或蛋白质混合物中功能性和反应性精氨酸残基的有效方法。
