CN110865188A

CN110865188A - Use of reagent for quantifying Khib in SLE

Info

Publication number: CN110865188A
Application number: CN201911023148.6A
Authority: CN
Inventors: 戴勇; 周献青; 汤冬娥; 刘冬舟; 洪小平; 徐慧璇
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2020-03-06
Also published as: WO2021077612A1

Abstract

The invention provides an application of a reagent for quantifying lysine 2-hydroxyisobutyryl modification level in protein in preparing a reagent for diagnosing and/or prognosing SLE. The invention carries out systematic and deep research on SLE from the perspective of proteomics, firstly discusses the 2-hydroxyisobutyrylation maps of normal people and SLE patients in the population, finds that obvious differences exist in histone lysine 2-hydroxyisobutyrylation modification of the normal people and SLE patients, analyzes 2-hydroxyisobutyrylation sites with differential expression from the differences, and obtains key markers related to SLE, so that the key markers can be used as corresponding diagnostic markers.

Description

Use of reagent for quantifying Khib in SLE

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 SLE.

Background

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects multiple systems throughout the body, mostly in the age range of 15-45 years, with a prevalence rate of about one thousandth in our country, and the total number of people is as high as 100 or more ten thousand. SLE is clinically characterized by a complex and diverse repertoire of immune abnormalities involving multiple pathways, and is primarily characterized by the production of large numbers of heterogeneous autoantibodies against self-antigens in the cells or circulatory system. The antigen-antibody complex is deposited on end organs such as heart, joints, liver and kidney. Approximately 50-80% of patients with SLE will develop Lupus Nephritis (LN), the most severe manifestation of SLE.

At present, most SLE patients can obtain good curative effect by adopting a conventional treatment method, but the prognosis difficulty is high, the course of disease is repeated for a long time, and aggravation and remission are alternated, so that heavy economic burden is caused to families and society. In recent years, scholars at home and abroad have studied the pathogenesis of SLE from the basic to clinical level, from environmental infection to individuals, and from the whole to various levels such as cells, proteins, genes, epigenetic modifications, and the like. It is widely believed that the onset of SLE is related to both intrinsic factors such as genetics and extrinsic factors such as environment and drugs.

In recent years, development of histone modification, proteomics, genomics, metabonomics and related new technologies provides a new thinking model and research platform for research of SLE, and many scholars perform multiple related researches and obtain certain results. Literature studies indicate that SLE has a new breakthrough in basic and clinical studies, and not only some potential candidate markers are found, but also the pathogenesis of SLE is newly elucidated, but the exact etiology and pathogenesis of SLE have not been elucidated so far. Therefore, new research approaches are sought that potentially provide new perspectives and new ideas for understanding the pathogenesis of SLE.

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-hydroxyisobutyrate and SLE in order to obtain the application of the differentially expressed 2-hydroxyisobutyryl 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 SLE patients and normal people in the preparation of a reagent for the diagnosis and/or prognosis of SLE.

According to a first aspect of the present invention, there is provided the use of an agent 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 SLE.

The invention has the beneficial effects that:

the invention carries out systematic and deep research on SLE from the perspective of proteomics, firstly discusses the 2-hydroxyisobutyrylation maps of normal people and SLE patients in the population, finds that obvious differences exist in histone lysine 2-hydroxyisobutyrylation modification of the normal people and SLE patients, analyzes 2-hydroxyisobutyrylation sites with differential expression from the differences, and obtains key markers related to SLE, so that the key markers can be used as corresponding diagnostic markers.

According to an embodiment of the present invention, the protein is selected from at least one of HBA2 protein, HBB protein, PRDX2 protein, BLVRB protein, LTF protein, SPTA1 protein, CA1 protein.

According to the embodiment of the invention, the site of lysine 2-hydroxyisobutyration modification is selected from at least one of amino acid 40 of HBA2 protein, amino acid 8 of HBA2 protein, amino acid 12 of HBA2 protein, amino acid 18 of HBB protein, amino acid 83 of HBB protein, amino acid 60 of HBB protein, amino acid 9 of HBB protein, amino acid 26 of PRDX2 protein, amino acid 119 of PRDX2 protein, amino acid 135 of PRDX2 protein, amino acid 178 of BLVRB protein, amino acid 315 of LTF protein, amino acid 476 of LTF protein, amino acid 541 of SPTA1 protein, amino acid 1708 of SPTA1 protein, amino acid 1782 of SPTA1 protein, amino acid 35 of CA1 protein, amino acid 81 of CA1 protein, amino acid 157 of CA1 protein, amino acid 157.

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 HBA2 protein, HBB protein, PRDX2 protein, BLVRB protein, LTF protein, SPTA1 protein, CA1 protein, in the preparation of an agent for the diagnosis and/or prognosis of SLE.

Drawings

FIG. 1 is the functional enrichment analysis of proteins corresponding to differential modification sites based on up-regulation of 2-hydroxyisobutyrylation of GO.

FIG. 2 is a functional enrichment assay of GO-based differentially modified site-corresponding proteins with down-regulated 2-hydroxyisobutyrylation.

FIG. 3 is a functional enrichment assay of proteins corresponding to differentially modified sites based on upregulation of 2-hydroxyisobutyrylation by KEEG.

FIG. 4 is a functional enrichment assay of proteins corresponding to differential modification sites for the downregulation of 2-hydroxyisobutyrylation based on KEEG.

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

SLE patients and normal human peripheral blood leukocytes were selected in 6 cases each.

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 SwissProt Human (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 assays in SLE patients and Normal humans

A total of 3684 2-hydroxyisobutyration sites were identified among the 1036 proteins, of which 3159 sites of 897 proteins contained quantitative information. Significant differences were detected at 343 sites for 344 proteins in SLE patients and normal, including 156 sites with 220 proteins and 187 sites with 124 proteins. The majority of 897 2-hydroxyisobutyrylated modified proteins comprise 1-2 modification sites, some of which comprise 3-7 modification sites, and the minority of which comprises 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 the criterion, >1.5 is considered up-regulation and <1/1.5 is considered down-regulation. A comparison of the number of differentially modified sites in SLE patients and normal persons is shown in Table 1.

TABLE 1 summary of differentially modified sites

From the above results, it can be seen that the level of modification of lysine 2-hydroxyisobutyrylation of histones in SLE patients is significantly lower compared to normal humans.

The fold difference of partial differential modification sites is shown in Table 2

TABLE 2 proteins corresponding to differentially modified sites

2.2 functional Classification of proteins corresponding to sites of differential modification

The subcellular localization of the 2-hydroxyisobutyrated modification site to the corresponding protein is characteristic, with most of the up-regulated 2-hydroxyisobutyrated protein distributed in the cytoplasm (49%), nucleus (19%), mitochondria (10%) and extracellular region (9%). In contrast, most down-regulated proteins are distributed in the cytoplasm (42%), nucleus (30%), extracellular region (14%) and mitochondria (7%). The results show that there is no significant difference in the localization of up-and down-regulated 2-hydroxyisobutylated proteins.

The GO functional classification was evaluated based on the biological processes, molecular functions and cellular components of all 2-hydroxyisobutylated proteins. Within the biological process category, most up-regulated 2-hydroxyisobutylated proteins are associated with cellular, biological, monomeric and metabolic processes. In contrast, most downregulated proteins are associated with cellular processes, biological regulation, and metabolic processes. In cellular component classification, most up-regulated 2-hydroxyisobutylated proteins are associated with cellular components such as cells, organelles, extracellular regions, etc., while most down-regulated proteins are associated with cellular components such as cells, organelles, cell membranes, etc. In molecular function, most up-regulated 2-hydroxyisobutylated proteins are associated with molecular functions such as binding, catalytic activity, structural molecular activity, etc., while most down-regulated proteins are associated with molecular functions such as binding, catalytic activity, structural molecular activity, etc. 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. 1-4 are functional enrichment assays of proteins corresponding to differential modification sites based on up and down regulation of 2-hydroxyisobutyrylation of GO and KEEG. As shown in fig. 1 to 4:

functional enrichment analysis based on GO shows that up-regulated 2-hydroxyisobutyrylated protein is highly enriched in various cell components such as adhesive connection, anchoring connection, cell matrix junction and the like, and comprises various molecular functions such as specific binding of protein domains, structural composition of ribosomes, activated structural molecules and the like, and is positioned in various biological processes such as endoplasmic reticulum protein, actin cytoskeleton recombination, regulation of virus life cycle and the like (figure 1). The down-regulated 2-hydroxyisobutyrated protein is highly enriched in a plurality of cell components such as cytoplasmic ribosome, ribosomal subunit, cytoplasmic part and the like, and comprises a plurality of molecular functions such as ribosome structural composition, activated structural molecule, DNA combination and the like, and a plurality of biological processes such as ribosome large subunit biogenesis, virus gene expression, and localization to endoplasmic reticulum protein and the like (figure 2).

Functional enrichment analysis based on KEGG showed that up-regulated 2-hydroxyisobutyrated protein was enriched only in 6 processes including ribosomes (figure 3). The down-regulated 2-hydroxyisobutyrylated protein was enriched in 3 processes, including ribosome, nitrogen metabolism, etc., which may be closely related to the complications of SLE (fig. 4).

Based on the above results, these 2-hydroxyisobutyrated modified proteins and corresponding modification sites are potential biomarkers for non-invasive diagnosis of SLE.

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 an agent 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 SLE.

2. The use according to claim 1, wherein the protein is selected from at least one of the group consisting of HBA2 protein, HBB protein, PRDX2 protein, BLVRB protein, LTF protein, SPTA1 protein, and CA1 protein.

3. The use of claim 2, wherein the site of lysine 2-hydroxyisobutyration modification is selected from at least one of amino acid 40 of HBA2 protein, amino acid 8 of HBA2 protein, amino acid 12 of HBA2 protein, amino acid 18 of HBB protein, amino acid 83 of HBB protein, amino acid 60 of HBB protein, amino acid 9 of HBB protein, amino acid 26 of PRDX2 protein, amino acid 119 of PRDX2 protein, amino acid 135 of PRDX2 protein, amino acid 178 of BLVRB protein, amino acid 315 of LTF protein, amino acid 476 of LTF protein, amino acid 541 of SPTA1 protein, amino acid 1708 of SPTA1 protein, amino acid 1782 of SPTA1 protein, amino acid 35 of CA1 protein, amino acid 81 of CA1 protein, amino acid 157 of CA1 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 an agent for quantifying the expression level of a protein, wherein the protein is selected from at least one of the group consisting of HBA2 protein, HBB protein, PRDX2 protein, BLVRB protein, LTF protein, SPTA1 protein, CA1 protein, for the preparation of an agent for the diagnosis and/or prognosis of SLE.

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