Proteomic analysis of protein lysine 2-hydroxyisobutyrylation (K_hib) in soybean leaves

Protein lysine 2-hydroxyisobutyrylation (K_hib) is a novel post-translational modification (PTM) discovered in cells or tissues of animals, microorganisms and plants in recent years. Proteome-wide identification of K_hib-modified proteins has been performed in several plant species, suggesting that K_hib-modified proteins are involved in a variety of biological processes and metabolic pathways. However, the protein K_hib modification in soybean, a globally important legume crop that provides the rich source of plant protein and oil, remains unclear.

In this study, the K_hib-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 K_hib-modified proteins was performed. Our results showed that a total of 4251 K_hib 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 K_hib-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 K_hib 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 K_hib site. Furthermore, 16 highly-connected clusters of K_hib proteins were retrieved from the global PPI network, which suggest that K_hib modifications tend to occur in proteins associated with specific functional clusters.

These findings suggest that K_hib 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 K_hib proteomic data obtained in this study will help to further elucidate the regulatory mechanisms of K_hib modification in soybean in the future.