Proteomics screening of PBMCs from IgAN patients and NCs
We carried out the experimental procedure in 9 steps (Fig. 1a) and established a library of proteins differentially expressed between IgAN patients and NCs. Altogether, 6502 DEPs were identified based on 52362 unique peptides. The confident identification of proteins required an FDR < 0.01 and at least two unique peptides per protein to be present; therefore, 5848 proteins met the above criteria. To check the acquired MS data, we verified that the mass error was between -11 and 4 ppm, which meets the requirement of mass accuracy (Fig. 1b). Most peptides ranged from 7 to 20 amino acids in length, which is consistent with the basic principle of trypsin digestion. The cutoff for the identification of DEPs or differentially expressed sites (DESs) was a P value < 0.05. A greater than 1.5-fold change was termed upregulation, while a fold change of <1/1.5 was termed downregulation for DEPs or DESs. Of the DEPs between IgAN patients and NCs, 111 were upregulated and 83 were downregulated (Fig. 1c), including LCN2 (IgA/NC ratio: 13.503, IgA/NC P value < 0.001), LTF (IgA/NC ratio: 13.304, IgA/NC P value < 0.001), MMP9 (IgA/NC ratio: 9.663, IgA/NC P value < 0.001), MPO (IgA/NC ratio: 5.675, IgA/NC P value < 0.001), S100A9 (IgA/NC ratio: 5.663, IgA/NC P value < 0.001), S100A8 (IgA/NC ratio: 5.079, IgA/NC P value < 0.001) and MYH9 (IgA/NC ratio: 1.571, IgA/NC P value < 0.001).
Functional classification of subcellular localization and GO analysis of the Khib proteome
The subcellular localization of the Khib proteome was illustrated by WoLF PSORT software, and the upregulated 2-hydroxyisobutyrylated proteins were found to be mainly distributed in the extracellular space (35%), cytoplasm (33%), nucleus (12%), plasma membrane (10%), and mitochondria (5%). On the contrary, the majority of the downregulated proteins were distributed in the cytoplasm (40%), nucleus (27%), and extracellular region (18%). A significant difference was not observed in the localization of the upregulated and downregulated proteins based on the data presented in our study.
To assess the 2-hydroxyisobutyrylated protein landscape in IgAN patients, the GO category (2nd Level) functional classification was assessed for all 2-hydroxyisobutyrylated proteins in three categories. Most of the upregulated 2-hydroxyisobutyrylated proteins were observed to be associated with the biological processes categories of single-organism process, cellular process, response to stimulus biological regulation, localization, and immune system process. Additionally, most of the DEPs were classified into the cell, organelle, and extracellular regions based on the cellular component category. In the molecular function classification, most of the DEPs were found to be involved in binding and catalytic activity (Fig. 2a). However, the majority of the downregulated proteins are involved in the single-organism process and cellular process categories; the cell, organelle, extracellular region, membrane and macromolecular complexes within the cellular component category; and binding, catalytic activity, and molecular function regulation within the molecular function classification (Fig. 2b). KOG category analysis was also performed through an NCBI database comparison. GO or KOG category functional classification revealed no significant difference in the DEPs between IgAN patients and NCs.
GO, KEGG, and protein domain functional enrichment of the Khib proteome
We further performed a GO, KEGG pathway, and protein domain enrichment analysis with the intention of assessing whether there was a significant DEP enrichment trend in some functional categories. The upregulated DEPs were markedly enriched in the cellular component of secretory granule, secretory vesicle, cytoplasmic vesicle, and intracellular vesicle (Fig. 3a) and in the biological processes of secretion, secretion by cell, response to bacteria, and antimicrobial humoral response (Fig. 3b). Furthermore, they were identified as being enriched in the molecular functions of fatty acid derivative binding, eicosatetraenoic acid-binding, arachidonic acid-binding, and RAGE receptor binding (Fig. 3c). In contrast, there was no significant GO functional enrichment of any cellular component, biological process or molecular function for the downregulated DEPs (Fig. 3d, Fig. 3e, Fig. 3f).
The KEGG-based functional enrichment analysis showed that the upregulated DEPs were significantly increased in 3 processes, including the hsa04614 renin-angiotensin system (Fig. S11), hsa04145 phagosome, and hsa04657 IL-17 signaling pathway, which may have an important association with IgAN (Fig. 4a). There were other enriched KEGG functional pathways. For example, the upregulated DEPs in hsa05418 fluid shear stress and atherosclerosis may be associated with IgAN complications (such as the occurrence of cardiovascular disease), and those in hsa05134 legionellosis and hsa05140 leishmaniasis along with hsa05146 amoebiasis may be associated with the activation of IgAN pathophysiology. The downregulated DEPs were enriched in hsa04217 necroptosis (Fig. 4b).
Furthermore, the domains of the upregulated DEPs were only significantly enriched in the S100/CaBP−9k−type calcium-binding subdomain, as shown in Fig. 4c. The protein domains enriched in the downregulated DEPs included globin/protoglobin, globin-like, globin, and SH3 domain (Fig. 4d).
Protein-protein interaction (PPI) network of the Khib proteins
To broaden the landscape of the cellular processes regulated through 2-hydroxyisobutyrylation in IgAN patients, we constructed a PPI network model of the Khib proteins (Fig. 5a). DEPs were compared by the STRING protein–protein interaction database, and the top 50 tightly linked protein pairs were visually presented by the R package “networkD3” tool only when their confidence scores were greater than 0.7 (high confidence), identifying a varied cellular function landscape of 2-hydroxyisobutyrylated proteins in IgAN patients. Evidently, S100A8, MPO, MMP9, ITGAM, ITGB2 and BPI had the most interactions with other proteins (Fig. 5b). For instance, we found direct relationships between P14780 (MMP9) and other DEPs, along with P05164 (MPO; Fig. 5c). It seems that the physiological links among these 2-hydroxyisobutyrylated protein complexes contribute to their cooperation and coordination in IgAN patients.
PRM-based validation
Eight proteins were quantified and confirmed by PRM analysis based on the results of TMT labeling quantitative analysis to verify the results of the whole-cell proteomics (Table 2). These proteins were selected according to their functional significance in the Khib proteome analysis in this study, and the ratio of protein abundance varied within a range. The identified proteins were quantified by PRM if at least 3 unique peptides were identified in each alternative protein. The PRM and TMT labeling quantification results were generally consistent, especially for the three following proteins: MPO (P05164), S100A9 (P06702) and S100A8 (P05109).
Proteome-wide analysis of Khib in PBMCs between IgAN patients and NCs
In this study, we performed a Khib analysis in PBMCs from IgAN patients and NCs, which has not been reported previously. To ensure the high confidence of the results, we filtered the identification data using the criteria of localization probability > 0.75, FDR < 0.01 and score > 40. The effect of protein expression on the modified signal was removed by normalization to that of the protein quantitation group, and the normalized data were used for the subsequent bioinformatics analysis. In total, 3684 Khib sites distributed across 1036 proteins were identified, and 3132 sites on 894 proteins were quantifiable. The majority of the 2-hydroxyisobutyrylated proteins carried 1 to 4 Khib sites. Approximately 6% of the proteins carried more than 10 Khib sites (Fig. 6a), of which 94 Khib-modified sites were present in MYH9, being the greatest number of identified Khib sites on a single 2-hydroxyisobutyrylated protein.
Table 2 The comparison of the quantification results between the TMT label and PRM methods.
Protein Accession
|
Protein Name
|
IgA Raletive Abundance
|
NC Raletive Abundance
|
IgA/NC Ratio(PRM)
|
IgA/NC Ratio(TMT)
|
P05164
|
MPO
|
1.48
|
0.52
|
2.82
|
5.68
|
P06702
|
S100A9
|
1.71
|
0.29
|
5.80
|
5.66
|
P05109
|
S100A8
|
1.63
|
0.37
|
4.36
|
5.08
|
P62753
|
RPS6
|
0.97
|
1.03
|
0.94
|
1.54
|
P63104
|
YWHAZ
|
0.72
|
1.28
|
0.56
|
0.74
|
P05106
|
ITGB3
|
0.68
|
1.32
|
0.52
|
1.02
|
P14625
|
HSP90B1
|
0.56
|
1.44
|
0.39
|
0.83
|
P05556
|
ITGB1
|
0.47
|
1.53
|
0.31
|
1.07
|
Identification of whole-cell Khib-modified proteins
In total, 122 Khib-modified proteins containing 171 sites with increased Khib modification and 168 Khib-modified proteins containing 257 specific sites were detected in the Khib DESs in IgAN patients (Fig. 6b). Among these DESs, we found that MYH9 mentioned above had 12 DESs, all of which had reduced Khib modification.
GO functional classification of Khib -modified proteins and their subcellular localization
In the subcellular localization of Khib-modified proteins in this study, most of the upregulated Khib-modified proteins were distributed in the cytoplasm (49%), nucleus (25%), and mitochondria (7%). In addition, the majority of the downregulated Khib-modified proteins were located in the cytoplasm (42%) and nucleus (26%). According to the data, no significant differences were observed in the subcellular localization of the DEPs.
To acquire the Khib-modified proteome landscape in IgAN patients, GO functional classification was performed for Khib-modified proteins depending on the associated biological processes, molecular functions, and cellular components. Most of the upregulated Khib-modified proteins were associated with cellular processes, biological regulation, single-organism process, and metabolic process within the biological processes category. However, most of the downregulated proteins were associated with cellular processes and single-organism processes. Within the cellular component classification, the upregulated 2-hydroxyisobutyrylated proteins, for the most part, were associated with cell, organelle, macromolecular complex, extracellular region, membrane and membrane-enclosed lumen. In addition, most of the downregulated proteins were associated with cell, organelle, membrane, extracellular region, macromolecular complex and membrane-enclosed lumen. Moreover, within the molecular function category, most of the upregulated DEPs were associated with binding, catalytic activity, structural molecule activity, and molecular function regulator. In contrast, most of the downregulated DEPs were associated with binding, catalytic activity, structural molecule activity, and molecular function regulator. There were no significant differences in the GO functional classification of the DEPs, implying that Khib modification may have wide-ranging biological functions.
GO, KEGG, and protein domain functional enrichment of the Khib-modified proteins
The upregulated 2-hydroxyisobutyrylated proteins were predominantly clustered in the cellular component of intracellular ribonucleoprotein complex, and ribonucleoprotein complex; molecular functions including structural constituent of ribosomes, structural molecule activity, peroxiredoxin activity and actin-binding; and biological processes including viral gene expression, nucleic acid metabolic process, cellular macromolecule biosynthetic process, viral life cycle and peptide biosynthetic process (Fig. 7). The downregulated 2-hydroxyisobutyrylated proteins were obviously clustered in the cellular component of cell membrane projection, membrane ruffles, leading edge membrane; molecular functions including cytoskeletal protein binding, actin binding and cell adhesion molecule binding; and biological processes including nuclear division, homotypic cell-cell adhesion and mitotic nuclear division.
KEGG-based functional enrichment analysis illustrated that the upregulated 2-hydroxyisobutyrylated proteins were enriched in processes (as shown in Fig. 8a) including ribosomes and the cell cycle. The downregulated 2-hydroxyisobutyrylated proteins were enriched in 16 processes, including regulation of the actin cytoskeleton, IL-17 signaling pathway, PI3K-Akt signaling pathway, fluid shear stress and atherosclerosis, and ribosome and phagosome, which have important associations with IgAN complications (Fig. 8b).
Furthermore, upregulated 2-hydroxyisobutyrylated proteins were enriched in domains such as nucleic acid-binding, OB-fold; nucleotide-binding alpha-beta plait domain; K homology domain, type 1; K homology domain; translation protein SH3-like domain; FERM, N-terminal; zinc finger, PHD-type; zinc finger, PHD-finger; rubredoxin-type fold; heat shock chaperonin-binding and FERM domain, demonstrating the competing and unique roles of Khib-modified proteins. Downregulated 2-hydroxyisobutyrylated proteins were observed to be enriched in protein domains including histidine kinase-like ATPase, C-terminal domain, integrin domain, heat shock protein Hsp90, N-terminal, calponin homology domain, ribosomal protein S5 domain 2-type fold; S100/CaBP-9k-type, calcium-binding, subdomain, EF-hand domain pair and FERM, N-terminal.
GO, KEGG, and protein domain cluster analyses
Based on the analyses of DEPs in the different comparison groups for the GO classification, KEGG pathway and protein domain enrichment, we applied cluster analyses to research the correlation between the function of DEPs in the comparison groups.
For the GO analysis cellular component, there was no enrichment in upregulated 2-hydroxyisobutyrylated proteins, whereas the downregulated 2-hydroxyisobutyrylated proteins were mainly enriched in neuromuscular junction, cell projection, cytoskeletal part, cytoskeleton, plasma membrane part, plasma membrane region, filopodium and membrane ruffles (Fig. S1 and S2).
The biological process enrichment of 2-hydroxyisobutyrylation was carried out and upregulated 2-hydroxyisobutyrylated proteins tended to be enriched in cellular functions such as biosynthetic process. We found that the downregulated proteins were mainly associated with homotypic cell-cell adhesion, animal organ development, response to fungus, integrin-mediated signaling pathway, regulation of plasma membrane organization and spindle organization (Fig. S3 and S4).
Moreover, the upregulated proteins were mainly clustered in the structural constituent of ribosomes, structural molecule activity, peroxiredoxin activity and various protein binding processes. Within the category of molecular function, the downregulated 2-hydroxyisobutyrylated proteins were specifically involved in the cellular binding process (Fig. S5 and S6).
The KEGG enrichment analysis of all DEPs identified the PI3K-Akt signaling pathway. Accordingly, the upregulated 2-hydroxyisobutyrylated proteins present in the most prominent enriched pathways were associated with the ribosome, cell cycle, and vitamin digestion and absorption, whereas the downregulated proteins were enriched in the ribosome and phagosome; diseases such as Staphylococcus aureus infection, pathogenic Escherichia coli infection, hypertrophic cardiomyopathy (HCM) and fluid shear stress and atherosclerosis; cellular signaling pathways such as the IL-17 signaling pathway and the PI3K-Akt signaling pathway, and cellular metabolism (Fig. S7 and S8).
The protein domains of the 2-hydroxyisobutyrylated proteins were evaluated. There were extremely upregulated enriched genes containing heat shock chaperonin-binding, rubredoxin-type fold, nucleic acid-binding, OB-fold and K homology domains. The highly downregulated proteins were enriched in the integrin domain; S100/CaBP-9k-type, calcium-binding, subdomain; ribosomal protein S5 domain 2-type fold; histidine kinase-like ATPase, C-terminal domain and heat shock protein Hsp90, N-terminal domain, similar to the results from functional classification of the protein domains (Fig. S9 and S10).