Biomarker for diagnosing systemic lupus erythematosus and application thereof

Abstract

The invention provides a biomarker for diagnosing systemic lupus erythematosus and application of the biomarker, and belongs to the field of biological medicine. The biomarker is at least one of the following five protein N-glycopeptides, specifically, IgG1N299-HexNAc (3), HexA (4), HexA (5), HexA (6), HexA (7), HexA (8) and HexA (8), f2N121-HexNAc (2), HexA (12), F2N121-HexNAc The invention relates to a C3N85-HexNAc (2) and Hex (1). (2), HexNAc (4), VTNN169-HexNAc (2) and And (1) APOBN1523-HexNAc (3), Hex (4) NeuAc (1). The five protein N-glycopeptides provided by the invention are used as biomarkers for SLE disease diagnosis, have the advantages of high accuracy, high sensitivity and high specificity, and provide new targets for diagnosis, intervention improvement and the like of SLE patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to biomarkers for diagnosing systemic lupus erythematosus and applications thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple systems, with a complex pathogenesis and highly heterogeneous clinical manifestations. Current diagnostic criteria suffer from insufficient sensitivity and specificity, making early or atypical cases prone to missed or misdiagnosis. In terms of laboratory testing, serological markers such as antinuclear antibodies (ANA), anti-double-stranded DNA antibodies (anti-dsDNA), and anti-Sm antibodies have some diagnostic value, but some patients test negative or their titers fluctuate, making it difficult to meet the needs of precise diagnosis and treatment.

[0003] In recent years, glycosylation, a key type of post-translational modification of proteins, has been shown to be closely associated with the development and progression of autoimmune diseases. Abnormal N-glycosylation can contribute to the immune disorders of systemic lupus erythematosus (SLE) by affecting immune cell function, antigen presentation, and the secretion of inflammatory factors. However, precise and specific N-glycopeptide biomarkers for the diagnosis of SLE have yet to be developed.

[0004] Therefore, there is an urgent need for biomarkers with high sensitivity and specificity for the diagnosis of SLE. Summary of the Invention

[0005] In view of this, the present invention aims to provide biomarkers for diagnosing systemic lupus erythematosus and applications thereof, aiming to solve at least one technical problem in the background technology.

[0006] The present invention is achieved in that: The first aspect of the present invention provides a biomarker for diagnosing systemic lupus erythematosus, wherein the biomarker is at least one of the following five protein N-glycopeptides; The five protein N-glycopeptides are specifically: IgG1_N299-HexNAc(3)Hex(4); F2_N121-HexNAc(2)Hex(12); C3_N85-HexNAc(2)Hex(1); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1); Among them, IgG1 is a subtype of serum immunoglobulin G, and its amino acid sequence is shown in SED ID NO.1; F2 is prothrombin, and its amino acid sequence is shown in SED ID NO.2; C3 is complement C3, and its amino acid sequence is shown in SED ID NO.3; VTN is vitronectin, and its amino acid sequence is shown in SED ID NO.4; APOB is apolipoprotein B-100, and its amino acid sequence is shown in SED ID NO.5; N is an amino acid residue, and the number after N is the site; HexNAc is a hexosamine residue, Hex is a hexose residue, and NeuAc is a sialic acid residue. The numbers in brackets represent the number of corresponding monosaccharide residues.

[0007] Furthermore, the biomarker is a combination of at least three of the five protein N-glycopeptides.

[0008] Furthermore, the biomarker is a combination of at least four of the five protein N-glycopeptides.

[0009] Furthermore, the biomarker is the entire combination of the five protein N-glycopeptides.

[0010] A second aspect of the present invention provides use of a reagent for detecting the expression level of the above-mentioned biomarker for diagnosing systemic lupus erythematosus in the preparation of a product for diagnosing systemic lupus erythematosus.

[0011] Furthermore, the product detects the expression level of the biomarker in the blood based on mass spectrometry.

[0012] Furthermore, the product is a kit or a reagent.

[0013] A third aspect of the present invention provides a product for diagnosing systemic lupus erythematosus, which comprises a reagent for detecting the expression level of the above-mentioned biomarker for diagnosing systemic lupus erythematosus.

[0014] Furthermore, the product detects the expression level of the biomarker in the blood based on mass spectrometry.

[0015] Furthermore, the product is a kit or a reagent.

[0016] This study identified five protein N-glycopeptide biomarkers with significant expression differences between blood samples from SLE patients and healthy controls. These five protein N-glycopeptides, alone or in combination, are proposed as biomarkers for SLE diagnosis, demonstrating high accuracy, sensitivity, and specificity, providing new targets for diagnosis and interventional improvement in SLE patients. The development of corresponding auxiliary early diagnostic reagents and kits based on these five protein N-glycopeptide biomarkers, alone or in combination, has broad scientific research value and clinical application, significantly facilitating early screening, clinical diagnosis, and therapeutic intervention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a boxplot of the differential expression of five protein N-glycopeptides in the blood between the SLE group and the HC group; Figure 2 The ROC curves of 5 protein N-glycopeptides as biomarkers in SLE group and HC group; Figure 3 The ROC curves of any two combinations of the five protein N-glycopeptides as biomarkers in the SLE group and the HC group are shown; Figure 4 The ROC curves of any three combinations of the five protein N-glycopeptides as biomarkers in the SLE group and the HC group; Figure 5 The ROC curves of any four combinations of the five protein N-glycopeptides as biomarkers in the SLE group and the HC group; Figure 6 Figure 2 is the ROC curve of the combination of all five protein N-glycopeptides as biomarkers in the SLE group and the HC group. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] 1. Experimental Subjects 138 patients with systemic lupus erythematosus were selected as the SLE group, and 58 healthy subjects without systemic lupus erythematosus were selected as the HC group. Their characteristics are shown in Table 1.

[0020] Table 1

[0021] Among them, some patients with systemic lupus erythematosus lack clinical information on indicators such as urine occult blood, urine protein, anti-double-stranded DNA antibodies, antinuclear antibodies, and anti-Sm antibodies. Therefore, the calculation of the positive proportion of the above indicators is based on the total number of patients with clinical information on the indicator.

[0022] 2. Screening of differentially expressed protein N-glycopeptides 1. Protein extraction The blood samples of the experimental subjects were taken out from -80℃, thawed in ice, and centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube and the protein concentration was determined using a BCA kit.

[0023] 2. Enzymatic hydrolysis (1) Equal amounts of protein from each blood sample were enzymatically hydrolyzed, and the sample volumes were adjusted to a consistent level using 8 M urea lysis buffer; (2) Dithiothreitol (DTT) was added to each sample to a final concentration of 5 mM and reduced at 56°C for 30 min; (3) Iodoacetamide (IAM) was added to each sample to a final concentration of 11 mM and incubated for 15 min at room temperature in the dark. (4) Transfer the alkylated sample to a 10 kD ultrafiltration tube and centrifuge at 12,000 g for 20 min at room temperature. Discard the waste liquid after ultrafiltration. (5) Add 300 μL of 8 M urea to the ultrafiltration tube, centrifuge at 12,000 g for 30 min at room temperature, and discard the waste liquid after ultrafiltration; repeat this step once more; (6) Add 200 μL of 20 mM ammonium bicarbonate to the ultrafiltration tube, centrifuge at 12,000 g for 20 min at room temperature, discard the ultrafiltration waste, and repeat this step twice; (7) Add 300 μL of trypsin at a ratio of 1:50 (protease:protein, m / m) to the ultrafiltration tube and incubate at 37°C overnight; (8) Centrifuge at 12000g for 30 min at room temperature to recover the peptide solution, then add 200 μL ddH2O to the ultrafiltration tube, centrifuge at 12000g for 20 min at room temperature to recover the peptide once, and combine the two peptide solutions for later use.

[0024] 3. Desalination (1) The peptide solution after enzymatic hydrolysis was acidified to pH 2-3 with 10% trifluoroacetic acid (TFA), centrifuged at 12000g for 10 min at room temperature, and the supernatant was transferred to a new EP tube; (2) Add 1 mL of anhydrous methanol to the SPE column for activation; (3) Add 1 mL of 0.1% TFA to equilibrate the column and repeat this step once more; (4) Load the acidified peptide solution onto the SPE column; (5) Add 1 mL of 0.1% TFA to the SPE column for desalting and repeat this step three times. (6) Add 800 μL of 80% acetonitrile (ACN) to the SPE column for elution, collect the eluted peptides, and quantify the peptides using a BCA kit.

[0025] 4. Intact glycopeptide modification enrichment method (1) Dissolve the peptide in 200 μL of enrichment buffer (80% acetonitrile / 5% trifluoroacetic acid); (2) The dissolved peptides were centrifuged at 20,000 g for 5 min, and the supernatant was transferred to a hydrophilic (HILIC) microcolumn and centrifuged at 1,000 g for about 15 min to complete the enrichment; (3) The hydrophilic microcolumn was then washed three times with enrichment buffer and centrifuged at 500 g for 5 min. (4) Elute the glycopeptide using 0.1% trifluoroacetic acid, 50 mM ammonium bicarbonate solution, and 50% acetonitrile, respectively, centrifuge at 500 g for 5 min, collect the combined eluate, and freeze-dry under vacuum; (5) Finally, desalt according to the instructions of C18 ZipTips, freeze-dry under vacuum, and then provide for liquid chromatography-mass spectrometry analysis.

[0026] 5. Liquid chromatography-mass spectrometry analysis (1) The peptides were dissolved in liquid chromatography mobile phase A and separated using a Vanquish Neo ultra-high performance liquid chromatography system; Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an aqueous solution containing 0.1% formic acid and 80% acetonitrile; Liquid phase gradient setting: 0 min-0.75 min, 4.0% B; 0.75 min-0.90 min, 4.0% B-8.0% B; 0.90 min-1.35 min, 8.0% B-8.5% B; 1.35 min-20.85 min, 8.5% B-22.5% B; 20.85 min-31.35 min, 22.5% B-35% B; 31.35 min-31.95 min, 35.0% B-55.0% B; 31.95 min-32.70 min, 55.0% B-99.0% B; 32.70 min-34.00 min, 99.0% B; the flow rate was maintained at 400 nl / min; (2) After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the NSI ion source for ionization and then entered the Orbitrap Astral mass spectrometer for analysis. The ion source voltage was set to 1900 V, the peptide parent ions were detected and analyzed using the Orbitrap detector, and the secondary fragment ions were detected and analyzed using the Astral detector. The primary mass spectrometer scan range was set to 700 m / z-2000 m / z, and the scan resolution was set to 240,000; the secondary mass spectrometer scan range had a fixed starting point of 120 m / z, and the secondary scan resolution was set to 80,000. The data acquisition mode used the data-dependent scanning (DDA) program, and the cycle time was set to 0.6 s. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 100%, the signal threshold was set to 25,000 ions / s, the maximum injection time was set to 5 ms, and the dynamic exclusion time of the tandem mass spectrometer scan was set to 15 s to avoid repeated scanning of the parent ions.

[0027] 6. Database Search Secondary mass spectrometry data were searched using MSFragger (v3.4). The search parameters were as follows: the database was Homo_sapiens_9606_SP_20230103.fasta (20,389 sequences), with a reverse library added to account for the false positive rate (FDR) due to random matches; the enzyme digestion method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum peptide length was set to 7 amino acid residues; the maximum number of peptide modifications was set to 3; and the mass error tolerance for primary precursor ions and secondary fragment ions was set to 20 ppm. Cysteine alkylation (Carbamidomethyl (C)) was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were variable modifications. Mass offsets were set to the glycosylation modification list. The FDR for protein and PSM identification was set to 1%.

[0028] 3. Verify diagnostic efficiency Figure 1 Figure 2 shows a comparison of the expression levels of five N-glycopeptides in the blood of SLE and HC subjects. Statistical tests and regression analysis were used to evaluate the differences in the expression levels of the five N-glycopeptides in the blood of the two groups, as well as the relationship between N-glycosylation patterns and clinical parameters. The diagnostic performance was assessed using receiver operating characteristic (ROC) curves. A diagnostic indicator with an area under the ROC curve (AUC) ≥ 0.9 was considered "highly accurate," an indicator with an AUC of 0.8 ≤ AUC < 0.9 was considered "accurate," and an indicator with a moderate accuracy of 0.7 ≤ AUC < 0.8 was considered "moderately accurate."

[0029] The 5 protein N-glycopeptides include: IgG1_N299-HexNAc(3)Hex(4); F2_N121-HexNAc(2)Hex(12); C3_N85-HexNAc(2)Hex(1); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1); Among them, IgG1 is a subtype of serum immunoglobulin G, and its amino acid sequence is shown in SED ID NO.1; F2 is prothrombin, and its amino acid sequence is shown in SED ID NO.2; C3 is complement C3, and its amino acid sequence is shown in SED ID NO.3; VTN is vitronectin, and its amino acid sequence is shown in SED ID NO.4; APOB is apolipoprotein B-100, and its amino acid sequence is shown in SED ID NO.5; N is an amino acid residue, and the number after N is the site; HexNAc is a hexosamine residue, Hex is a hexose residue, and NeuAc is a sialic acid residue. The numbers in brackets represent the number of corresponding monosaccharide residues.

[0030] The above five protein N-glycopeptides were used alone or in combination as serum diagnostic markers to distinguish between SLE and HC groups. The ROC curve results of SLE and HC were as follows: Figures 2 to 6 The AUC, sensitivity, and specificity are shown in Tables 2 to 6.

[0031] Table 2

[0032] Table 3

[0033] Table 4

[0034] Table 5

[0035] Table 6

[0036] Depend on Figure 1 It can be seen that the expression levels of IgG1_N299-HexNAc(3)Hex(4), F2_N121-HexNAc(2)Hex(12), C3_N85-HexNAc(2)Hex(1), VTN_N169-HexNAc(2)Hex(4), and APOB_N1523-HexNAc(3)Hex(4)NeuAc(1) in the blood of systemic lupus erythematosus (SLE) patients were higher than those in healthy controls (HC), and the differences were very significant (p less than 0.001).

[0037] Figure 2 The results in Table 2 showed that the above differentially expressed protein N-glycopeptides could effectively distinguish SLE from HC alone (AUC>0.8), but the sensitivity and specificity needed to be improved.

[0038] like Figures 3 to 6As shown in Tables 3 to 6, any combination of two, three, four, or all of the five protein N-glycopeptides can effectively distinguish SLE from HC as biomarkers, and the effect is significantly better than that of a single protein N-glycopeptide. Among them, the advantages of the three-combination, the four-combination, and the all-combination are more obvious, with AUC ≥ 0.97, sensitivity ≥ 93.5%, and specificity ≥ 93.1%. Some combinations have AUC = 1, and the sensitivity and / or specificity is 100%.

[0039] Experimental results demonstrate that five protein N-glycosylated peptides can serve as blood diagnostic biomarkers to distinguish healthy individuals from those with SLE, and can be used to develop diagnostic products for systemic lupus erythematosus, such as kits and reagents. This biomarker combination demonstrates enhanced sensitivity, specificity, and accuracy, providing important evidence for further clinical research and offering new insights into the diagnosis and treatment of SLE.

[0040] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A biomarker for diagnosing systemic lupus erythematosus, characterized in that: The biomarker is at least one of the following five protein N-glycopeptides; The five protein N-glycopeptides are specifically: IgG1_N299-HexNAc(3)Hex(4); F2_N121-HexNAc(2)Hex(12); C3_N85-HexNAc(2)Hex(1); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1); Among them, IgG1 is a subtype of serum immunoglobulin G, and its amino acid sequence is shown in SED ID NO.1; F2 is prothrombin, and its amino acid sequence is shown in SED ID NO.2; C3 is complement C3, and its amino acid sequence is shown in SED ID NO.3; VTN is vitronectin, and its amino acid sequence is shown in SED ID NO.4; APOB is apolipoprotein B-100, and its amino acid sequence is shown in SED ID NO.5; N is an amino acid residue, and the number after N is the site; HexNAc is a hexosamine residue, Hex is a hexose residue, and NeuAc is a sialic acid residue. The numbers in brackets represent the number of corresponding monosaccharide residues.

2. The biomarker for diagnosing systemic lupus erythematosus according to claim 1, characterized in that The biomarker is a combination of at least three of the five protein N-glycopeptides.

3. The biomarker for diagnosing systemic lupus erythematosus according to claim 2, characterized in that: The biomarker is a combination of at least four of the five protein N-glycopeptides.

4. The biomarker for diagnosing systemic lupus erythematosus according to claim 3, characterized in that The biomarker is the entire combination of the five protein N-glycopeptides.

5. Use of a reagent for detecting the expression level of the biomarker for diagnosing systemic lupus erythematosus according to any one of claims 1 to 4 in the preparation of a product for diagnosing systemic lupus erythematosus.

6. The use according to claim 5, characterized in that The product detects the expression level of the biomarker in the blood based on mass spectrometry.

7. The use according to claim 6, characterized in that The product is a kit or a reagent.

8. A product for diagnosing systemic lupus erythematosus, characterized in that: The product comprises a reagent for detecting the expression level of the biomarker for diagnosing systemic lupus erythematosus according to any one of claims 1 to 4.

9. The product for diagnosing systemic lupus erythematosus according to claim 8, characterized in that: The product detects the expression level of the biomarker in the blood based on mass spectrometry.

10. The product for diagnosing systemic lupus erythematosus according to claim 8, characterized in that: The product is a kit or a reagent.

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