CN110726845A

CN110726845A - Use of reagent for quantifying Khib in IgA nephropathy

Info

Publication number: CN110726845A
Application number: CN201911022610.0A
Authority: CN
Inventors: 戴勇; 黄少颖; 汤冬娥; 郑凤屏; 张欣洲
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2020-01-24

Abstract

The invention provides an application of a reagent for quantifying lysine 2-hydroxyisobutyryl modification level in protein in preparation of a reagent for diagnosis and/or prognosis of IgA nephropathy. The invention carries out systematic and deep research on IgAN from the view point of proteomics, discusses the 2-hydroxyisobutyrylation maps of normal people and IgAN patients in the crowd for the first time, finds that obvious difference exists in histone lysine 2-hydroxyisobutyrylation modification of the normal people and the IgAN patients, and analyzes 2-hydroxyisobutyrylation sites with differential expression to obtain key markers related to the IgAN, so that the key markers can be used as corresponding diagnosis markers.

Description

Use of reagent for quantifying Khib in IgA nephropathy

Technical Field

The invention relates to the technical field of immune diseases, in particular to application of a quantitative lysine 2-hydroxyisobutyryl modified reagent in IgA nephropathy.

Background

IgA nephropathy (IgAN) is a group of immunopathological diagnosis names, which was first reported by j.berger and n.hindglaisll in 1968, also called Berger disease, glomerulonephritis caused by various causes, a series of clinical and pathological changes are caused by the fact that IgA or an immune complex mainly containing IgA is deposited on the glomerular mesangial area or capillary wall in a limited or diffused manner in different degrees under immunofluorescence or immunohistochemistry, the most common cause of glomerular hematuria is the most common primary glomerular disease in the world at present, accounts for 10-40% of all renal biopsy pathologies, accounts for 20-50% of the primary glomerular disease, and is the highest in asian areas and the most common glomerular disease in China, and is one of the main causes of end-stage renal disease (ESRD).

IgAN is better in young and strong years, and more men than women. The etiology and pathogenesis of the disease are not completely clear, and related researches indicate that IgAN is a complex disease determined by multiple genes and multiple factors. Clinical manifestations are various, the clinical manifestations mainly take repeated macroscopic hematuria or microscopic hematuria as the first symptom, and can accompany proteinuria of different degrees, and the clinical manifestations are various, and almost all the clinical characteristics of glomerulonephritis are covered. The diagnosis depends on the kidney biopsy pathology, the pathological manifestations mainly include mesangial proliferative glomerulonephritis, and the pathological degrees are different. The Gutierrez E study showed that macroscopic hematuria may induce IgAN acute renal failure, with up to 25% of patients with acute renal failure with macroscopic hematuria failing to recover to basal renal function levels. The YamagataK study showed that proteinuria patients older than 40 years are more likely to develop renal insufficiency. Furthermore, the prognosis is also different. Data show that 40% of IgA patients require months to years from asymptomatic to end stage renal disease.

The pathogenesis of IgAN is not fully elucidated at present, and immunoregulatory abnormalities are the focus and focus of current research. The epigenetic mechanism plays an important role in the normal development and the function of an immune system, and the influence of external factors causes the epigenetic mechanism to generate unbalance in immune response, which can cause abnormal expression of genes and immune dysfunction, thereby causing the occurrence of autoimmune diseases. Two major mechanisms of epigenetics are DNA methylation and histone modification, which in turn have important effects on chromatin structure alteration, gene transcription and replication.

Post-translational modification of proteins is an important means by which cells regulate protein function and life processes. Post-translational modifications (PTMs) of proteins may alter the properties of the protein by altering the covalent bonds by proteolytic means or by adding modifying groups to one or more amino acids. Based on the DNA coding capacity, we predicted that the number of post-translational modifications greatly exceeded the number of proteins. More than 200 different types of post-translational modifications have now been found that dynamically regulate various biological events such as subcellular localization, protein degradation, protein-protein interactions, conformational changes, signal transduction, and gene transcription.

Post-translational modification of histones, an important aspect of epigenetics, is an important way for cells to regulate gene transcription. The same post-translational modification can occur at different histone sites, and the same histone site can undergo different post-translational modifications, and these post-translational modifications of histones and the interrelationship between the post-translational modifications constitute the "histone code".

In recent years, with advances in mass spectrometry technology, new post-translational modifications of proteins have been discovered, including new sites and new modification types. Among these, acylation modifications of protein lysine residues, i.e., lysine acylation, are considered to be one of the most widely distributed post-translational modifications involving gene transcription, energy metabolism, and signaling. Recent studies on lysine acylation have also made a dramatic progress in the past 5 years, thanks to the development of these studies by mass spectrometry and pan-antibody enrichment. In species, in addition to lysine acetylation, new histone acylation modifications are continually being discovered, such as propionylation, butyrylation, malonylation, succinylation, crotonylation, 2-hydroxyisobutyrylation, and the like. To date, these novel post-translational modifications of lysine have attracted considerable attention in the biological field, and the study of these newly discovered modified proteomics and enzymes would greatly facilitate our understanding of the function and regulation of these modifications.

Recently, lysine 2-hydroxyisobutyrylation (Khib), a newly discovered histone post-translational modification, is considered as a novel histone marker in eukaryotic cells, and is widely distributed in the biological metabolic pathway of prokaryotes. Lysine 2-hydroxyisobutyrylation was first identified in human and mouse histones as an important epigenetic mechanism on nucleosomal histones during chromatin kinetic recombination. This histone mark was initially preliminarily identified by Mass Spectrometry (MS), which was further validated by chemical and biochemical methods. Previous animal studies have shown that lysine 2-hydroxyisobutyryl modification of histones is involved in sperm cell differentiation and plays a crucial role in the regulation of chromatin function. This modification is well conserved during evolution and by analyzing the substrate proteins in biological processes of translation, protein degradation and energy metabolism, we have found that this modification is widely distributed in various biological metabolic pathways and plays a role in transcription. Acetylation, methylation of histone lysine have been extensively studied, but recent findings suggest that histone lysine 2-hydroxyisobutyrylation is structurally and functionally distinct from the former two modifications, with unique chemical structure, specific genomic distribution, and different kinetic profiles between model systems.

Therefore, it is necessary to search the correlation between the modified 2-hydroxyisobutyrophenol and IgAN in order to obtain the application of the differentially expressed 2-hydroxyisobutyrylated modification to the preparation of corresponding diagnostic or prognostic reagents.

Disclosure of Invention

The technical problem to be solved by the invention is how to provide an application of a reagent for quantifying 2-hydroxyisobutyryl modified differential expression of an IgAN patient and a normal person in the preparation of a reagent for the diagnosis and/or prognosis of IgAN.

According to a first aspect of the present invention, the present invention provides the use of an agent for quantifying the level of lysine 2-hydroxyisobutyration modification in proteins for the preparation of a diagnostic and/or prognostic agent for IgAN.

The invention has the beneficial effects that:

the invention carries out systematic and deep research on IgAN from the view point of proteomics, discusses the 2-hydroxyisobutyrylation maps of normal people and IgAN patients in the crowd for the first time, finds that obvious difference exists in histone lysine 2-hydroxyisobutyrylation modification of the normal people and the IgAN patients, and analyzes 2-hydroxyisobutyrylation sites with differential expression to obtain key markers related to the IgAN, so that the key markers can be used as corresponding diagnosis markers.

According to an embodiment of the invention, the Protein is selected from at least one of Lactotransferrin Protein, Annexin A3 Protein, Myeloperoxoxidase Protein, Protein S100-A9 Protein, Protein S100-A8 Protein, Histone H4 Protein, Lysozyme C Protein, synthetic membrane Protein VAT-1homolog Protein, Annexin A1 Protein, Myosin-9 Protein.

According to the embodiment of the invention, the site of lysine 2-hydroxyisobutyryl modification is selected from the group consisting of amino acid 282 of lactotranferin Protein, amino acid 315 of lactotranferin Protein, amino acid 638 of lactotranferin Protein, amino acid 154 of Annexin 3 Protein, amino acid 169 of Annexin A3 Protein, amino acid 556 of Myelopoxidase Protein, amino acid 38 of Protein S100-A9 Protein, amino acid 106 of Protein S100-A9 Protein, amino acid 84 of Protein S100-A8 Protein, amino acid 85 of Protein S100-A8 Protein, amino acid 92 of Protein S100-A8 Protein, amino acid 13 of Protein Histone H4 Protein, amino acid 32 of Protein H4 Protein, amino acid 4 of Protein H60, amino acid 8692 of Protein Synstone H4, amino acid D245 of Protein C115 Protein C1-amino acid log of Protein, amino acid D1 of Protein A3651-A3655 Protein, amino acid D of Protein H4 Protein, amino acid D, At least one of 287 th amino acid of Annexin A1 protein, 180 th amino acid of Myosin-9 protein, 202 nd amino acid of Myosin-9 protein, 228 th amino acid of Myosin-9 protein, 1024 th amino acid of Myosin-9 protein, 1441 th amino acid of Myosin-9 protein and 1775 th amino acid of Myosin-9 protein.

According to an embodiment of the invention, the protein is derived from peripheral blood mononuclear cells, preferably leukocytes.

According to a second aspect of the present invention, there is provided use of an agent for quantifying the expression level of a Protein selected from at least one of Lactotransferrin Protein, Annexin A3 Protein, Myeloperoxoxidase Protein, Protein S100-A9 Protein, Protein S100-A8 Protein, Histone H4 Protein, lysozym C Protein, synthetic vein membrane Protein VAT-1homolog Protein, Annexin 1 Protein, and Myosin-9 Protein, in the preparation of a diagnostic and/or prognostic agent for IgAN.

Drawings

FIGS. 1-2 show the subcellular localization of the 2-hydroxyisobutyryl modification site for the corresponding protein. Wherein FIG. 1 is a graph showing the distribution of up-regulated subcellular localization in 2-hydroxyisobutylated protein, and FIG. 2 is a graph showing the distribution of down-regulated subcellular localization in 2-hydroxyisobutylated protein.

FIGS. 3-4 are the GO functional classes of proteins corresponding to the 2-hydroxyisobutyryl modification sites evaluated. Wherein, FIG. 3 is the distribution result of GO functional classification in up-regulated 2-hydroxyisobutylated protein, and FIG. 4 is the distribution result of GO functional classification in down-regulated 2-hydroxyisobutylated protein.

FIGS. 5-12 are statistical distributions of differentially expressed proteins based on 2-hydroxyisobutyrylation modification of GO and KEEG. Wherein, FIG. 5 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO upregulation in cellular components, and FIG. 6 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO downregulation in cellular components. Fig. 7 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO up-regulation in biological processes, and fig. 8 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO down-regulation in biological processes. FIG. 9 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO upregulated in molecular function, and FIG. 10 is a statistical distribution of 2-hydroxyisobutyrylated modified differentially expressed proteins based on GO downregulated in molecular function. FIG. 11 is an enrichment distribution result of up-regulated 2-hydroxyisobutyrylated modified differentially expressed protein in KEGG pathway, and FIG. 12 is an enrichment distribution result of down-regulated 2-hydroxyisobutyrylated modified differentially expressed protein in KEGG pathway.

FIGS. 13-16 are cluster analysis results based on functional enrichment of differentially modified sites of 2-hydroxyisobutyrylation of GO and KEEG. Wherein, fig. 13 is a cluster analysis result of functional enrichment of 2-hydroxyisobutyrated modified sites based on up-regulation of GO, and fig. 14 is a cluster analysis result of functional enrichment of 2-hydroxyisobutyrated modified sites based on down-regulation of GO. FIG. 15 is a cluster analysis result of functional enrichment of 2-hydroxyisobutyryl modification sites based on upregulation of KEGG, and FIG. 16 is a cluster analysis result of functional enrichment of 2-hydroxyisobutyryl modification sites based on downregulation of KEGG.

Detailed Description

The conception, the specific structure, and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below, so that the objects, the features, and the effects of the present invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

1. Research method

1.1 sample selection and protein extraction:

6 cases of peripheral blood leukocytes of IgAN patients and normal persons were confirmed by renal biopsy.

Samples were removed from-80 ℃ and lysed by sonication with 4 volumes of lysis buffer (8M urea, 1% protease inhibitor, 3. mu. MTSA, 50mM NAM and 2mM EDTA) separately. Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to remove cell debris, and the supernatant was transferred to a new centrifuge tube and subjected to protein concentration measurement using the BCA kit.

1.2 enzymatic hydrolysis of pancreatin

Dithiothreitol was added to the protein solution to give a final concentration of 5mM, and the solution was reduced at 56 ℃ for 30 min. After that, iodoacetamide was added to give a final concentration of 11mM, and incubated for 15min at room temperature in the absence of light. Finally the urea concentration of the sample was diluted to below 2M. Adding pancreatin in a mass ratio of 1:50 (pancreatin: protein), and performing enzymolysis at 37 ℃ overnight. Adding pancreatin in a mass ratio of 1:100 (pancreatin: protein), and continuing enzymolysis for 4 h.

1.3TMT markers

The pancreatin peptide fragments were desalted with Strata X C18(Phenomenex) and vacuum freeze-dried. The peptide fragment was dissolved at 0.5MTEAB and labeled according to the protocol of the TMT kit. The simple operation is as follows: thawing the labeled reagent, dissolving with acetonitrile, mixing with the peptide segment, incubating at room temperature for 2h, mixing the labeled peptide segment, desalting, and vacuum freeze drying.

1.4HPLC fractionation

The peptide fragments were fractionated by high pH reverse phase HPLC using an Agilent 300 extended C18 column (5 μm size, 4.6mm inner diameter, 250mm length). The operation is as follows: the peptide fragment grading gradient is 8-32% acetonitrile, the pH value is 9, 60 components are separated within 60min, then the peptide fragments are combined into 18 components, and the combined components are subjected to vacuum freeze drying and then subjected to subsequent operation.

1.5 liquid chromatography-Mass Spectrometry coupled analysis

The peptide fragment was dissolved in mobile phase A (0.1% (v/v) formic acid aqueous solution) by liquid chromatography, and then separated by using EASY-nLC 1000 ultra performance liquid system. The mobile phase A is an aqueous solution containing 0.1 percent of formic acid and 2 percent of acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Setting a liquid phase gradient: 0-19 min, 7-22% of B; 19-32 min, 22-35% of B; 32-36 min, 35-80% B; 36-40 min, 80% B, and the flow rate is maintained at 800 nL/min.

The peptide fragments were separated by ultra high performance liquid system, injected into NSI ion source for ionization and then analyzed by QOxective Plus mass spectrometry. The ion source voltage was set at 2.0kV and both the peptide fragment parent ion and its secondary fragment were detected and analyzed using the high resolution Orbitrap. The scanning range of the primary mass spectrum is set to 350-1800m/z, and the scanning resolution is set to 70000; the fixed starting point of the secondary mass spectrum scanning range is 100m/z, and the Orbitrap scanning resolution is set to 17500. The data acquisition mode uses a data-dependent scanning (DDA) program, namely, after the primary scanning, the first 20 peptide fragment parent ions with the highest signal intensity are selected to sequentially enter an HCD collision cell for fragmentation by using 30% of fragmentation energy, and secondary mass spectrometry is also sequentially performed. To improve the effective utilization of the mass spectra, the Automatic Gain Control (AGC) was set to 5E4, the signal threshold was set to 10000ions/s, the maximum injection time was set to 200ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scans of parent ions.

1.6 database search

Secondary mass spectral data were retrieved using Maxquant (v1.5.2.8). And (3) retrieval parameter setting: the database is SwissProtHuman (20130 sequences), a reverse library is added to calculate false positive rate (FDR) caused by random matching, and a common pollution library is added to the database and is used for eliminating the influence of pollution protein in the identification result; the enzyme cutting mode is set as Trypsin/P; the number of missed cutting sites is set to 2; the minimum length of the peptide segment is set to be 7 amino acid residues; the maximum modification number of the peptide fragment is set to be 5; the First-level parent ion mass error tolerance of the First search and the Main search is respectively set to be 20ppm and 5ppm, and the mass error tolerance of the second-level fragment ions is 0.02 Da. Cysteine alkylation is set as fixed modification, and variable modification is oxidation of methionine and acetylation of the N-terminal of the protein. The quantitative method is set as TMT-6plex, and the FDR of protein identification and PSM identification is set as 1%.

1.7 Mass Spectrometry quality control detection

First, we examined the mass shift (mass error) of all identified peptide fragments. The mass error is centered at 0 and is concentrated in the range of less than 10ppm, which indicates that the mass error is satisfactory. Secondly, the length of the vast majority of peptide fragments is distributed between 8-20 amino acid residues, which accords with the rule of pancreatin digestion of peptide fragments, and indicates that the sample preparation reaches the standard.

2. Results of the experiment

2.1 proteomic and 2-hydroxyisobutyrylation analysis of IgAN patients and Normal humans

3684 2-hydroxyisobutyrylated modification sites in 1036 proteins were identified, of which 3159 sites of 897 proteins contained quantitative information. Among the differentially expressed proteins detected in IgAN patients and normal persons, 111 proteins were up-regulated and 83 proteins were down-regulated. Among the differential modification sites, a significant difference was detected at 428 sites for 290 proteins, including a significant upregulation at 171 sites for 122 proteins and a significant downregulation at 257 sites for 168 proteins. The majority of 897 2-hydroxyisobutyrylated modified proteins contained 1-2 modification sites, and the minority included 8 or more modification sites. Most peptides vary in length from 8 to 20 amino acids, following the rule of trypsin digestion.

With a 1.5 fold change threshold as standard, >1.5 is considered up-regulation, <1/1.5 is considered down-regulation, P-value < 0.05. The comparison of the amount of differentially modified proteins in IgAN patients and normal humans is shown in Table 1:

TABLE 1 summary of differential modified proteins for IgAN/Normal control

From the above results, it can be seen that the histone from IgAN patients has significantly higher level of lysine 2-hydroxyisobutyryl-modified protein compared to normal human. The fold-difference of the partial differential modified proteins is shown in table 2.

TABLE 2 differential modification sites and corresponding proteins for IgAN patients

2.2 functional Classification of proteins corresponding to sites of differential modification

The subcellular localization of the 2-hydroxyisobutyrated modification site corresponding protein was characteristic, with most of the up-regulated 2-hydroxyisobutyrated protein distributed in the extracellular region (35%), cytoplasm (33%), nucleus (12%), plasma membrane (10%) and mitochondria (5%) (fig. 1). In contrast, most of the down-regulated proteins were distributed in cytoplasm (40%), nucleus (27%), extracellular region (18%) and mitochondria (8%) (fig. 2). The results show that there is no significant difference in the localization of up-and down-regulated 2-hydroxyisobutylated proteins.

GO annotations fall into 3 major classes: biological processes (Biological processes), cell composition (cellular component) and Molecular Function (Molecular Function), elucidate the Biological role of proteins from different perspectives. The GO functional classification was evaluated based on the biological processes, molecular functions and cellular components of all 2-hydroxyisobutylated proteins (fig. 3, fig. 4). Within the biological process category, most up-regulated 2-hydroxyisobutylated proteins are associated with monomeric processes, cellular processes, responses to stimuli, responses to biological stimuli, sites, immune system processes belonging to the category of biological processes. In contrast, most down-regulated proteins are associated with monomeric, cellular processes. In cellular component classification, most up-regulated 2-hydroxyisobutylated proteins are associated with cells, organelles and extracellular regions, while most down-regulated proteins are associated with cells, organelles, extracellular regions, cell membranes and macromolecular complexes. In molecular function, most up-regulated 2-hydroxyisobutylated proteins are associated with protein binding and catalytic activity, while most down-regulated proteins are associated with modulators of 2-hydroxyisobutylated protein binding, catalytic activity and molecular function. The GO functional classification showed no significant difference between up-and down-regulation of 2-hydroxyisobutylated protein. This result indicates that 2-hydroxyisobutyrylation may have a wide range of biological functions.

2.3 functional enrichment analysis of proteins corresponding to differentially modified sites

FIGS. 5-12 are functional enrichment assays of protein 5 corresponding to differential modification sites based on up and down regulation of 2-hydroxyisobutyrylation of GO and KEEG. As shown in fig. 5 to 12:

functional enrichment analysis based on GO showed that up-regulated 2-hydroxyisobutyrylated protein was mainly enriched in secretory granules, secretory vesicles, cytoplasmic vesicles and intracellular vesicles in terms of cellular composition (fig. 5), and down-regulated 2-hydroxyisobutyrylated protein was extensive and not significantly enriched (fig. 6). In terms of biological processes, up-regulated 2-hydroxyisobutyrylated protein was mainly enriched in secretions, cellular secretion, response to bacteria, antimicrobial humoral response (fig. 7), and down-regulated 2-hydroxyisobutyrylated protein was broader, without significant enrichment (fig. 8). In terms of molecular function, up-regulated 2-hydroxyisobutyrylated protein was mainly enriched in fatty acid derivative binding, eicosatetraenoic acid binding, arachidonic acid binding, RAGE receptor binding (fig. 9), and down-regulated 2-hydroxyisobutyrylated protein was broader, yet not significantly enriched (fig. 10).

Functional enrichment analysis based on KEGG showed that up-regulated 2-hydroxyisobutyrated protein was enriched only in 3 possible processes associated with IgAN, respectively: renin-angiotensin system, phagosome, IL-17 signaling pathway (fig. 11). The down-regulated 2-hydroxyisobutyrylated protein was mainly enriched in necrotic apoptosis (FIG. 12).

2.42 Cluster analysis of differentially modified sites by hydroxyisobutyration

FIGS. 13-16 are cluster analysis results of functional enrichment of differentially modified sites based on up and down regulation of 2-hydroxyisobutyrylation by GO and KEEG. As shown in fig. 13 to 16:

GO enrichment cluster analysis (fig. 13, fig. 14) shows that in bioprocess classification, up-regulated 2-hydroxyisobutyration differential modification sites are highly enriched in the cellular macromolecule biosynthesis process, while down-regulated 2-hydroxyisobutyration differential modification sites are mostly related to negative regulation of the biosynthesis process, nuclear division, spindle organization, response to fungi, and integrin-mediated signaling pathway. Enrichment analysis of cellular component taxonomy revealed that only a few of the up-regulated 2-hydroxyisobutyrylated differentially modified sites were associated with ribonucleoprotein complexes, while for down-regulated sites, they were associated with neuromuscular junctions, cell projections, cytoskeleton and plasma membranes. Within the molecular functional class, up-regulated 2-hydroxyisobutyration differential modification sites are primarily associated with peroxidase activity, and down-regulated 2-hydroxyisobutyration differential modification sites are primarily associated with phospholipase a2 inhibitor activity, phospholipase inhibitor activity, calcium-dependent phospholipid binding, and various molecular binding including tubulin binding, extracellular matrix binding, and protein kinase binding.

KEGG is an information network that links known intermolecular interactions. Enrichment cluster analysis based on KEGG showed that the most significant enrichment pathway among up-regulated 2-hydroxyisobutyryl differential modification sites was related to ribosome, cell cycle, vitamin digestion and absorption (fig. 15). In contrast, PI3K-AKT signaling pathway, IL-17 signaling pathway, phagosome and toxoplasma associated diseases, fluid shear stress and atherosclerosis associated diseases, Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), Dilated Cardiomyopathy (DCM), Hypertrophic Cardiomyopathy (HCM), leishmaniasis, pathogenic e.coli infections, and pertussis are the most significant pathways for the expression of down-regulated sites of differential modification of 2-hydroxyisobutyration (fig. 16).

The quantitative proteome data showed that levels of Lactotransferrin Protein, Annexin A3 Protein, Myeloperoxoxidase Protein, Protein S100-A9 Protein, Protein S100-A8 Protein, Histone H4 Protein, Lysozyme C Protein, synthetic vein membrane Protein VAT-1homolog Protein, Annexin A1 Protein, Myosin-9 Protein were significantly up-regulated in peripheral blood leukocytes of IgAN patients compared to the control group. Of these significantly up-regulated proteins, most 2-hydroxyisobutyration modification sites are significantly down-regulated, including: amino acid 282 of Lactotransferrin Protein, amino acid 315 of Lactotransferrin Protein, amino acid 638 of Lactotransferrin Protein, amino acid 154 of Annexin A3 Protein, amino acid 169 of Annexin A3 Protein, amino acid 556 of Myeloperoxoxidase Protein, amino acid 38 of Protein S100-A9 Protein, amino acid 106 of Protein S100-A9 Protein, amino acid 84 of Protein S100-A8 Protein, amino acid 85 of Protein S100-A8 Protein, amino acid 92 of Protein S100-A8 Protein, amino acid 13 of Protein Histone H4 Protein, amino acid 32 of Protein Histone H4 Protein, amino acid 60 of Protein Histone H4 Protein, amino acid 92 of Protein H4 Protein, amino acid 115 of Protein C Protein, amino acid 115 of Protein Synthesin T295 Protein, amino acid 287-369 of Protein, amino acid 23 of Protein A1 Protein, amino acid 287-1 Protein, amino acid 287 of Protein A369 Protein, Amino acid 202 of Myosin-9 protein, amino acid 228 of Myosin-9 protein, amino acid 1024 of Myosin-9 protein, amino acid 1441 of Myosin-9 protein, and amino acid 1775 of Myosin-9 protein. Based on the above results, these 2-hydroxyisobutyrylated modified proteins and corresponding modification sites may be potential biomarkers for non-invasive diagnosis of IgAN.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. Use of a reagent for quantifying the level of lysine 2-hydroxyisobutyration modification in a protein for the preparation of a reagent for the diagnosis and/or prognosis of IgA nephropathy.

2. The use according to claim 1, wherein said Protein is selected from at least one of Lactotransferrin Protein, Annexin 3 Protein, myoperoxoxidase Protein, Protein S100-A9 Protein, Protein S100-A8 Protein, Histone H4 Protein, Lysozyme C Protein, synthetic vein membrane Protein VAT-1homolog Protein, Annexin A1 Protein, and Myosin-9 Protein.

3. The use according to claim 2, wherein the site of lysine 2-hydroxyisobutyryl modification is selected from the group consisting of amino acid 282 of lactotranferin Protein, amino acid 315 of lactotranferin Protein, amino acid 638 of lactotranferin Protein, amino acid 154 of Annexin A3 Protein, amino acid 169 of Annexin A3 Protein, amino acid 556 of Myeloperoxoxidase Protein, amino acid 38 of Protein S100-A9, amino acid 106 of Protein S100-A9 Protein, amino acid 84 of Protein S100-A8 Protein, amino acid 85 of Protein S100-A8 Protein, amino acid 92 of Protein S100-A8 Protein, amino acid 13 of Protein Histone H4 Protein, amino acid 32 of Protein H4 Protein, amino acid 60 of Protein H4 Protein, amino acid 4 of Protein Synstosoman H4, amino acid 115 of Protein C1-amino acid T1-glycoprotein, amino acid 1 of Protein C115, and amino acid T295 of Protein C1-D, At least one of 245 th amino acid of Annexin A1 protein, 287 th amino acid of Annexin A1 protein, 180 th amino acid of Myosin-9 protein, 202 th amino acid of Myosin-9 protein, 228 th amino acid of Myosin-9 protein, 1024 th amino acid of Myosin-9 protein, 1441 th amino acid of Myosin-9 protein and 1775 th amino acid of Myosin-9 protein.

4. The use according to any one of claims 1 to 3, wherein the protein is derived from peripheral blood mononuclear cells.

5. Use of a reagent for quantifying the expression level of a Protein for the preparation of a reagent for the diagnosis and/or prognosis of IgA nephropathy, wherein the Protein is at least one Protein selected from the group consisting of Lacotransferrin Protein, Annexin A3 Protein, Myeloperoxoxidase Protein, Protein S100-A9 Protein, Protein S100-A8 Protein, Histone H4 Protein, Lysozyme C Protein, synthetic vein membrane Protein VAT-1homolog Protein, Annexin A1 Protein, and Myosin-9 Protein.

6. The use of claim 5, wherein the protein is from peripheral blood mononuclear cells.