Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang)

Background Lysine 2-hydroxyisobutyrylation (K-hib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, K-hib modification has not been reported thus far in rhubarb. Results In this study, we performed the first global analysis of K-hib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping K-hib modification peptides matched on 1525 K-hib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these K-hib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the K-hib site, and a total of 13 K-hib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. Conclusions A total of 4333 K-hib-modified peptides on 1525 proteins were identified. The K-hib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GK(hib) and k.kkk horizontal ellipsis .K-hib horizontal ellipsis .kkkkk, were extracted from a total of 13 K-hib-modified peptides. This study provides comprehensive K-hib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of K-hib modification, and the K-hib proteome data will facilitate further investigations of the roles and mechanisms of K-hib modification in rhubarb.