蛋白质瓜氨酸化是在Ca2+存在下由肽基精氨酸脱亚胺酶(PAD)调节的不可逆的翻译后修饰过程。这一过程与自身免疫性疾病的发生发展密切相关,癌症,神经系统疾病,心脑血管疾病,和其他重大疾病。利用生物质光谱法分析瓜氨酸蛋白由于其丰度低而面临巨大挑战,缺乏亲和标签,质荷比变化小,以及对同位素和脱酰胺干扰的敏感性。常用的研究蛋白质瓜氨酸化的方法主要涉及对肽的鸟嘌呤侧链的脲基团进行化学衍生,以增加瓜氨酸化肽的质荷比差。然后引入富含亲和力的标记以通过质谱有效地提高蛋白质瓜氨酸化的灵敏度和准确性。2,3-丁二酮或苯乙二醛化合物通常用作衍生试剂,以增加瓜氨酸化肽的质荷比差异,并且观察到所得衍生物含有α-二羰基结构。迄今为止,然而,没有关于二羰基化合物与瓜氨酸肽反应性的相关研究报道。在这项研究中,我们使用基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)确定了六种α-二羰基和两种β-二羰基化合物是否与标准瓜氨酸化肽发生衍生化反应。在α-二羰基化合物中,2,3-丁二酮和乙二醛与几种标准瓜氨酸化肽有效反应,但产生了一系列副产品。苯乙二醛,甲基乙二醛,1,2-环己二酮,和1,10-菲咯啉-5,6-二酮也用标准瓜氨酸化肽有效衍生,生成单个导数。因此,确定了一种可以产生单一衍生物的新衍生方法。在β-二羰基化合物中,1,3-环己二酮和2,4-戊二酮成功地与标准瓜氨酸化肽反应,并生成了一个导数。然而,它们的反应效率非常低,表明β-二羰基化合物不适合于瓜氨酸化肽的化学衍生化。上述结果表明,α-二羰基结构对于实现瓜氨酸化肽的有效和特异性化学衍生是必需的。此外,α-二羰基结构的侧链决定了衍生物的结构,衍生效率,以及副产品的产生(或其他)。因此,可以通过合成含有亲和标记的α-二羰基结构化合物来实现瓜氨酸肽的特异性富集和精确鉴定。所提出的方法能够通过MS鉴定瓜氨酸化蛋白及其修饰位点,从而更好地了解瓜氨酸化蛋白在不同组织中的分布。该研究结果将有助于研究瓜氨酸化蛋白在多种疾病中的作用机制。
Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two β-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the β-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the β-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.