蛋白质瓜氨酸化是一种不可逆的翻译后修饰过程，在 Ca2+ 存在下受肽基精氨酸脱亚胺酶 (PAD) 调节。这一过程与自身免疫性疾病、癌症、神经系统疾病、心脑血管疾病等重大疾病的发生和发展密切相关。由于其丰度低、缺乏亲和标签、质荷比变化小以及易受同位素和脱酰胺干扰的影响，通过生物质光谱法分析蛋白质瓜氨酸化面临着巨大的挑战。研究蛋白质瓜氨酸化常用的方法主要是对肽鸟嘌呤侧链的脲基进行化学衍生化，以增加瓜氨酸肽的质荷比差。然后引入亲和力富集的标签，有效提高质谱分析蛋白质瓜氨酸化的灵敏度和准确性。 2,3-丁二酮或苯基乙二醛化合物常被用作衍生化试剂，以增加瓜氨酸肽的质荷比差异，并且观察到所得衍生物含有α-二羰基结构。然而迄今为止，还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。在本研究中，我们使用基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF MS) 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肽发生衍生反应。在α-二羰基化合物中，2,3-丁二酮和乙二醛可以与几种标准瓜氨酸肽有效反应，但会产生一系列副产物。苯乙二醛、甲基乙二醛、1,2-环己二酮和 1,10-菲咯啉-5,6-二酮也可以用标准瓜氨酸肽有效衍生，生成单一衍生物。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-戊二酮成功与标准瓜氨酸肽反应，并生成单一衍生物。然而，它们的反应效率非常低，表明β-二羰基化合物不适合瓜氨酸肽的化学衍生化。上述结果表明，α-二羰基结构是实现瓜氨酸肽高效、特异化学衍生化所必需的。此外，α-二羰基结构的侧链决定了衍生物的结构、衍生化效率和副产物的产生（或其他）。因此，通过合成含有亲和标签的α-二羰基结构化合物，可以实现瓜氨酸肽的特异性富集和精确鉴定。该方法能够通过质谱鉴定瓜氨酸蛋白及其修饰位点，从而更好地了解瓜氨酸蛋白在不同组织中的分布。这些发现将有益于研究瓜氨酸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.