Use of biomarkers in the manufacture of a product for diagnosing systemic lupus erythematosus

Abstract

The application provides application of biomarkers in preparation of products for diagnosing systemic lupus erythematosus, and belongs to the field of biological medicine. The biomarkers include C3_N85-HexNAc(2)Hex(1); C3 is complement C3, the amino acid sequence of which is shown as SED ID NO.3; N is an amino acid residue, and the number after N is a site; HexNAc is a hexosamine residue, Hex is a hexose residue, NeuAc is a sialic acid residue, and the number in the bracket represents the number of corresponding monosaccharide residues. The biomarkers also include at least one of F2_N121-HexNAc(2)Hex(12), VTN_N169-HexNAc(2)Hex(4) and APOB_N1523-HexNAc(3)Hex(4)NeuAc(1). The application proposes that the above protein N-glycopeptide is used as a biomarker for diagnosing SLE, has the advantages of high accuracy, high sensitivity and high specificity, and provides a new target for diagnosis and intervention improvement of SLE patients.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202511245032.2, filed on September 2, 2025, with a priority date of July 15, 2025, entitled "Biomarkers for the diagnosis of systemic lupus erythematosus and their application". Technical Field

[0002] This invention belongs to the field of biomedical technology, and specifically relates to the application of biomarkers in the preparation of products for diagnosing systemic lupus erythematosus. Background Technology

[0003] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple systems. Its pathogenesis is complex, and its clinical manifestations are highly heterogeneous. Current diagnostic criteria suffer from insufficient sensitivity or specificity, especially in early or atypical cases, which are prone to being missed or misdiagnosed. Regarding laboratory testing, while serological markers such as antinuclear antibodies (ANA), anti-dsDNA antibodies, and anti-Sm antibodies have some diagnostic value, they are often negative or show fluctuating titers in some patients, making it difficult to meet the needs of precise diagnosis and treatment.

[0004] In recent years, glycosylation modification, as an important type of post-translational protein modification, has been proven to be closely related to the occurrence and development of autoimmune diseases. Among them, abnormal N-glycosylation can participate in the immune dysregulation of SLE by affecting immune cell function, antigen presentation, and inflammatory factor secretion. However, no precise and specific N-glycopeptide biomarkers for the diagnosis of SLE have yet been developed.

[0005] Therefore, there is an urgent need for biomarkers with high sensitivity and specificity that can be used to diagnose systemic lupus erythematosus. Summary of the Invention

[0006] Therefore, the present invention aims to provide the application of biomarkers in the preparation of products for diagnosing systemic lupus erythematosus, and to solve at least one of the technical problems in the background art.

[0007] This invention is implemented as follows: The first aspect of the present invention provides a biomarker for diagnosing systemic lupus erythematosus, said biomarker being 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 the 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 vitrin, 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, NeuAc is a sialic acid residue, and the number in parentheses represents the number of the corresponding monosaccharide residues.

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

[0009] Furthermore, the biomarker is a combination of at least four of the five protein N-glycopeptides.

[0010] Furthermore, the biomarker is the complete combination of the five protein N-glycopeptides.

[0011] A second aspect of the present invention provides the use of reagents for detecting the expression levels of the above-mentioned biomarkers for diagnosing systemic lupus erythematosus in the preparation of products for diagnosing systemic lupus erythematosus.

[0012] Furthermore, the product uses mass spectrometry to detect the expression level of the biomarker in the blood.

[0013] Furthermore, the product is a kit or reagent.

[0014] A third aspect of the present invention provides a product for diagnosing systemic lupus erythematosus, the product comprising reagents for detecting the expression levels of the aforementioned biomarkers for diagnosing systemic lupus erythematosus.

[0015] Furthermore, the product uses mass spectrometry to detect the expression level of the biomarker in the blood.

[0016] Furthermore, the product is a kit or reagent.

[0017] This invention identifies significant differences in the expression levels of five N-glycopeptide biomarkers in blood samples from SLE patients and healthy individuals. Therefore, it proposes these five N-glycopeptides, alone or in combination, as biomarkers for SLE diagnosis, offering advantages of high accuracy, high sensitivity, and high specificity, providing new targets for the diagnosis and intervention of SLE patients. The development of corresponding auxiliary early diagnostic reagents and kits based on these five N-glycopeptide biomarkers, alone or in combination, has broad research value and clinical applications, providing significant convenience for early screening, clinical diagnosis, and intervention treatment. Attached Figure Description

[0018] Figure 1 Box plot showing the differential expression of five N-glycopeptides in the blood of the SLE group and the HC group; Figure 2 ROC curves of five protein N-glycopeptides as biomarkers in the SLE and HC groups; Figure 3 ROC curves of any two combinations of five protein N-glycopeptides as biomarkers in the SLE and HC groups; Figure 4 ROC curves of any three combinations of five protein N-glycopeptides as biomarkers in the SLE and HC groups; Figure 5 ROC curves of any four combinations of five protein N-glycopeptides as biomarkers in the SLE group and HC group; Figure 6 ROC curves of all five protein N-glycopeptide combinations as biomarkers in the SLE and HC groups; Figure 7 To validate the box plots showing the differential expression of five N-glycopeptides in the blood of the SLE group and the HC group; Figure 8 To verify the ROC curves of five N-glycopeptides as biomarkers in the SLE and HC groups; Figure 9 To verify the ROC curves of any two combinations of the five N-glycopeptides from the set of proteins as biomarkers in the SLE group and HC group; Figure 10 To verify the ROC curves of any three combinations of the five N-glycopeptides from the set of proteins as biomarkers in the SLE group and HC group; Figure 11 To verify the ROC curves of any four combinations of the five N-glycopeptides from the set of proteins as biomarkers in the SLE group and HC group; Figure 12 To verify the ROC curves of the combined N-glycopeptides of the five proteins as biomarkers in the SLE and HC groups. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] I. Experimental Subjects The experimental set included 138 patients with systemic lupus erythematosus (SLE) as the SLE group and 58 healthy individuals without SLE as the HC group; the validation set included 51 patients with SLE as the SLE group and 42 healthy individuals without SLE as the HC group; the characteristic information is shown in Table 1 below.

[0021] Table 1

[0022] Some patients with systemic lupus erythematosus lack clinical information on indicators such as occult blood in urine, protein in urine, anti-double-stranded DNA antibody, antinuclear antibody, and anti-Sm antibody. Therefore, when calculating the positive rate of the above indicators, the total number of patients with clinical information on the above indicators is used as the benchmark.

[0023] II. Screening for differentially expressed protein N-glycopeptides 1. Protein extraction Blood samples from the subjects were taken out from -80℃, thawed on ice, centrifuged at 12000g for 10 min at 4℃ to remove cell debris, and the supernatant was transferred to a new centrifuge tube for protein concentration determination using a BCA kit.

[0024] 2. Enzymatic hydrolysis (1) Take equal amounts of protein from each blood sample for enzymatic digestion, and adjust the volume of the digested samples to be consistent using 8M urea lysis buffer; (2) Dithiothreitol (DTT) was added to each sample to a final concentration of 5 mM, and the sample was reduced at 56 °C for 30 min. (3) Iodoacetamide (IAM) was added to each sample to a final concentration of 11 mM, and the samples were alkylated and incubated at room temperature in the dark for 15 min. (4) Transfer the alkylated sample to a 10kD ultrafiltration tube, centrifuge at 12000g for 20min at room temperature, and discard the waste liquid after ultrafiltration; (5) Add 300 μL of 8M urea to the ultrafiltration tube, centrifuge at 12000g 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 12000 g for 20 min at room temperature, discard the waste liquid after ultrafiltration, and repeat this step twice. (7) Add 300 μL of trypsin to the ultrafiltration tube at a ratio of 1:50 (protease: protein, m / m) and incubate overnight at 37°C; (8) Centrifuge at 12000g for 30 min at room temperature to recover the peptide solution. Then add 200μL ddH2O to the ultrafiltration tube and centrifuge at 12000g for 20 min at room temperature to recover the peptide once. Combine the two peptide solutions for later use.

[0025] 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 the column to equilibrate, 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 to remove salt, and repeat this step 3 times; (6) Add 800 μL of 80% acetonitrile (ACN) to the SPE column for elution, collect the eluted peptides, and quantify the peptides using the BCA kit.

[0026] 4. Methods for enriching and modifying intact glycopeptides (1) Dissolve the peptide in 200 μL of enrichment buffer solution (80% acetonitrile / 5% trifluoroacetic acid); (2) After dissolving the peptide fragments, centrifuge at 20000g for 5 min, transfer the supernatant to a hydrophilic microcolumn, and centrifuge at 1000g for about 15 min to complete the enrichment; (3) Then wash the hydrophilic microcolumn three times with enrichment buffer and centrifuge at 500g for 5 min; (4) Elute the glycopeptides using 0.1% trifluoroacetic acid, 50mM ammonium bicarbonate solution and 50% acetonitrile respectively, centrifuge at 500g for 5min, collect and combine the eluents and freeze dry under vacuum; (5) Finally, desalted according to the C18 ZipTips instruction manual, vacuum freeze-dried and then used for liquid chromatography-mass spectrometry analysis.

[0027] 5. Liquid chromatography-mass spectrometry analysis (1) The peptide fragments were dissolved in liquid chromatography mobile phase A and then separated using the Vanquish Neo ultra-high performance liquid chromatography system; Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an aqueous solution containing 0.1% formic acid and 80% acetonitrile. Liquid gradient settings: 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; flow rate maintained at 400 nl / min. (2) After separation by an ultra-high performance liquid chromatography (UHPLC) system, the peptide fragments were injected into an NSI ion source for ionization and then analyzed by an Orbitrap Astral mass spectrometer. The ion source voltage was set to 1900V. The precursor peptide ion was detected and analyzed using an Orbitrap detector, and the secondary fragment ions were detected and analyzed using an Astral detector. The primary mass spectrometry scan range was set to 700m / z-2000m / z, and the scan resolution was set to 240000. The secondary mass spectrometry scan range had a fixed starting point of 120m / z, and the secondary scan resolution was set to 80000. The data acquisition mode used a data-dependent scanning (DDA) program, and the cycle time was set to 0.6s. To improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 100%, the signal threshold was set to 25000 ions / s, the maximum injection time was set to 5ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 15s to avoid repeated scanning of the precursor ion.

[0028] 6. Database search Secondary mass spectrometry data were searched using MSFragger (v3.4). Search parameter settings: the database was Homo_sapiens_9606_SP_20230103.fasta (20389 sequences), a reverse library was added to calculate the false positive rate (FDR) due to random matching; the restriction 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, with variable modifications including methionine oxidation and N-terminal acetylation of the protein. Mass offsets were set to the glycosylation modification list. The FDR for both protein identification and PSM identification was set to 1%.

[0029] III. Verifying Diagnostic Efficiency Figure 1 and Figure 7 The graphs show a comparison of the expression levels of five N-glycopeptides in the blood of SLE and HC subjects in the experimental and validation sets, respectively. Based on the differences in the expression levels of these five N-glycopeptides in the blood of SLE and HC subjects and the relationship between N-glycosylation characteristics and clinical parameters, statistical tests and regression analyses were used for evaluation. Diagnostic efficacy was evaluated using receiver operating characteristic (ROC) curves. A diagnostic indicator was considered "highly accurate" when the area under the ROC curve (AUC) ≥ 0.9, "accurate" when 0.8 ≤ AUC < 0.9, and "moderately accurate" when 0.7 ≤ AUC < 0.8.

[0030] The five 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 the 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 vitrin, 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, NeuAc is a sialic acid residue, and the number in parentheses represents the number of the corresponding monosaccharide residues.

[0031] The above five N-glycopeptides, alone or in combination, were used as serum diagnostic markers to distinguish between the SLE and HC groups. The ROC curve results for SLE and HC in the experimental set are shown below. Figures 2 to 6 As shown, the ROC curve results for SLE and HC in the validation set are presented. Figures 8 to 12 As shown, the AUC, sensitivity, and specificity of the experimental and validation sets are presented in Tables 2 to 6.

[0032] Table 2

[0033] Table 3

[0034] Table 4

[0035] Table 5

[0036] Table 6

[0037] Depend on Figure 1 and Figure 7 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 patients with systemic lupus erythematosus (SLE) are all higher than those in healthy individuals (HC), and the differences are highly significant (p < 0.001).

[0038] Figure 2 , Figure 8 The results in Table 2 show that the differentially expressed N-glycopeptides can effectively distinguish between SLE and HC on their own, but their sensitivity and specificity need to be improved.

[0039] like Figures 3 to 6 , Figures 9 to 12 As shown in Tables 3 to 6, any two, three, four, or all combinations of the five protein N-glycopeptides can effectively distinguish between SLE and HC as biomarkers, and the effect is significantly better than that of individual protein N-glycopeptides. Among them, the advantages of three, four, and all combinations are more obvious, with some combinations having AUC=1 and sensitivity and / or specificity of 100%.

[0040] Experimental results show that five 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 combination of biomarkers exhibits higher sensitivity, specificity, and accuracy. It provides important evidence for further clinical research and offers new insights into SLE diagnosis and treatment.

[0041] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of biomarkers in the preparation of products for diagnosing systemic lupus erythematosus, characterized in that, The biomarkers include C3_N85-HexNAc(2)Hex(1); Wherein, C3 is complement C3, and its amino acid sequence is shown in SED ID NO.3; N is an amino acid residue, and the number after N is the site; HexNAc is a hexosamine residue, Hex is a hexose residue, NeuAc is a sialic acid residue, and the number in parentheses represents the number of the corresponding monosaccharide residues.

2. The application according to claim 1, characterized in that, The biomarker also includes at least one of the following N-glycopeptides: F2_N121-HexNAc(2)Hex(12); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1); Among them, F2 is prothrombin, and its amino acid sequence is shown in SED ID NO.2; VTN is vitrin, 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.

3. The application according to claim 2, characterized in that, The biomarkers also include combinations of at least two of the following: F2_N121-HexNAc(2)Hex(12); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1).

4. The application according to claim 2, characterized in that, The biomarkers also include all combinations of F2_N121-HexNAc(2)Hex(12); VTN_N169-HexNAc(2)Hex(4); APOB_N1523-HexNAc(3)Hex(4)NeuAc(1).

5. The application according to claim 1, characterized in that, The product uses mass spectrometry to detect the expression levels of the biomarkers in blood.

6. The application according to claim 5, characterized in that, The product is a kit or reagent.

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