RESULTS: In this study, the Khib-modified proteins in soybean leaves were identified for the first time using affinity enrichment and high-resolution mass spectrometry-based proteomic techniques, and a systematic bioinformatics analysis of these Khib-modified proteins was performed. Our results showed that a total of 4251 Khib sites in 1532 proteins were identified as overlapping in three replicates (the raw mass spectrometry data are available via ProteomeXchange with the identifier of PXD03650). These Khib-modified proteins are involved in a wide range of cellular processes, particularly enriched in biosynthesis, central carbon metabolism and photosynthesis, and are widely distributed in subcellular locations, mainly in chloroplasts, cytoplasm and nucleus. In addition, a total of 12 sequence motifs were extracted from all identified Khib peptides, and a basic amino acid residue (K), an acidic amino acid residue (E) and three aliphatic amino acid residues with small side chains (G/A/V) were found to be more preferred around the Khib site. Furthermore, 16 highly-connected clusters of Khib proteins were retrieved from the global PPI network, which suggest that Khib modifications tend to occur in proteins associated with specific functional clusters.
CONCLUSIONS: These findings suggest that Khib modification is an abundant and conserved PTM in soybean and that this modification may play an important role in regulating physiological processes in soybean leaves. The Khib proteomic data obtained in this study will help to further elucidate the regulatory mechanisms of Khib modification in soybean in the future.
结果:在这项研究中,首次使用亲和富集和基于高分辨率质谱的蛋白质组学技术鉴定了大豆叶片中的Khib修饰蛋白,并对这些Khib修饰的蛋白质进行了系统的生物信息学分析。我们的结果显示,1532种蛋白质中的总共4251个Khib位点被鉴定为在三个重复中重叠(原始质谱数据可通过具有PXD03650标识符的ProteomeXchange获得)。这些Khib修饰的蛋白质参与了广泛的细胞过程,特别是富含生物合成,中心碳代谢和光合作用,并广泛分布在亚细胞位置,主要在叶绿体中,细胞质和细胞核。此外,从所有鉴定的Khib肽中提取了总共12个序列基序,和碱性氨基酸残基(K),发现酸性氨基酸残基(E)和具有小侧链的三个脂肪族氨基酸残基(G/A/V)更优选在Khib位点周围。此外,从全球PPI网络中检索到16个高度连接的Khib蛋白簇,这表明Khib修饰倾向于发生在与特定功能簇相关的蛋白质中。
结论:这些发现表明,Khib修饰是大豆中丰富而保守的PTM,并且这种修饰可能在调节大豆叶片的生理过程中起重要作用。本研究获得的Khib蛋白质组学数据将有助于进一步阐明大豆Khib修饰的调控机制。