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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Lysine 2-hydroxyisobutyrylation (Khib); Motif; Post-translational modification (PTM); Proteomics; Soybean.

Fig. 1

Global analysis of lysine 2-hydroxyisobutyrylation (K_hib) peptides and proteins identified in soybean leaves. A Mass error and score distribution of all identified K_hib-modified peptides. B Venn diagram shows the K_hib peptides identified in three experiments. C Venn diagram shows the K_hib proteins identified in three experiments. D Peptide length distribution of all K_hib-modified peptides. E Protein distribution of K_hib-modified sites contained in each protein

Fig. 2

Functional classification, secondary structure and subcellular localization analysis of the K_hib-modified proteins in soybean leaves. A Functional classification based on GO annotations. B Subcellular localization distribution of K_hib-modified proteins. C Predicted secondary structure of K_hib-modified peptides. D Predicted surface accessibility of K_hib-modified peptides

Fig. 3

Enrichment analysis of the K_hib-modified proteins in soybean leaves. A GO enrichment in terms of biological process, cellular component and molecular function. B KEGG pathway enrichment. The x-axis represents fold enrichment and the y-axis represents the categories of GO terms

Fig. 4

Representative KEGG pathways showing significant enrichment of K_hib-modified proteins in soybean leaves. A Central metabolism, including glycolysis/gluconeogenesis, pentose phosphate pathway, TCA cycle and oxidative phosphorylation. B Ribosome. The identified K_hib enzymes are indicated with a red background

Fig. 5

Motif analysis of the identified K_hib peptides in soybean leaves. A The sequence motifs of amino acids from − 10 to + 10 flanking the K_hib sites. The size of the letter represents the frequency of the amino acid residue residing at that position (B) Heat map analysis of the frequency of amino acid residues residing from -10 to + 10 positions around the K_hib sites. The enrichment or dispersion of amino acids are shown in red or green, respectively. C The histogram shows the number of K_hib-modified peptides matched for each motif.

Fig. 6

PPI network analysis of the K_hib-modified proteins in soybean leaves. Four representatives of PPI subnetworks, including ribosome, oxidative phosphorylation, photosynthesis and carbon fixation in photosynthetic organisms are presented