Method, system and kit for detecting retinopathy related proteome of premature infant

Abstract

The invention provides a method for detecting the expression quantity of retinopathy-related proteomes of premature infants. The method comprises the following steps: obtaining a tear sample from a premature infant, and obtaining a protein expression profile from the tear sample; detecting a retinopathy-related proteome in the protein representation spectrum, wherein the retinopathy-related proteome comprises one or more up-regulated proteins and down-regulated proteins; calculating the logarithmic double change value (Log2FC) of each up-regulated protein relative to the reference expression quantity of the up-regulated protein, and similarly calculating the logarithmic double change value of each down-regulated protein; a list is generated that includes up-regulated proteins having a log-doubled change value greater than or equal to a positive threshold value and down-regulated proteins having a log-doubled change value less than or equal to a negative threshold value.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 693,209, filed September 11, 2024, and U.S. Provisional Patent Application No. 63 / 767,518, filed March 5, 2025, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the fields of medicine and bioinformatics. More specifically, this invention relates to methods, systems, and kits for detecting proteomes associated with retinopathy of prematurity (ROP) in infants. Background Technology

[0003] Retinopathy of prematurity (ROP) is a serious ophthalmic disease characterized by abnormal proliferation of blood vessels in the retina of premature infants. This pathological neovascularization typically occurs at the junction of vascular and avascular areas in the immature retina and can further develop into fibrous scar tissue. Contraction of this fibrotic tissue can eventually lead to retinal detachment, resulting in permanent vision loss. Therefore, ROP is recognized globally as a leading cause of preventable childhood blindness. With advancements in neonatal care and increasing survival rates for premature infants, the global incidence of ROP has risen significantly. Despite its clinical relevance, the underlying etiology and pathophysiological mechanisms of ROP remain not fully understood.

[0004] Retinopathy of prematurity (ROP) progresses rapidly and severely, clinically classified into five stages: Stage 1 is the appearance of a slight boundary between the vascular and avascular retina; Stage 2 is the formation of ridges; Stage 3 is the occurrence of pathological neovascularization; Stage 4 is partial retinal detachment; and Stage 5 is complete retinal detachment. Furthermore, ROP can be divided into three concentric retinal zones centered on the optic disc based on the location and morphology of the lesions: Zone I is the innermost zone, encompassing the macula and optic disc; Zone II is the mid-peripheral retina; and Zone III is the crescent-shaped peripheral retina. Timely recognition of disease progression is crucial, as early intervention with anti-vascular endothelial growth factor (VEGF) therapy or laser photocoagulation can effectively prevent disease progression and maintain vision. Early detection of disease progression is extremely important because early intervention with VEGF therapy or laser photocoagulation can control the disease and protect vision.

[0005] Currently, indirect fundus examination is considered the gold standard for screening retinopathy of prematurity (ROP) in clinical practice. However, this method requires specialized equipment and is highly dependent on the subjective interpretation of experienced ophthalmologists. Therefore, different clinicians will inevitably interpret subtle retinal changes differently, and their diagnoses of disease staging may also differ, often leading to inconsistencies. Furthermore, the examination process usually requires repeated pupil dilation using cycloplegic or mydriatic agents, which can place a physiological burden on vulnerable preterm infants and cause side effects. Although imaging-based ROP screening methods exist, their prognostic capabilities are limited; they typically only detect the disease after it has occurred and cannot provide effective prediction or early risk assessment. Consequently, many preterm infants do not receive timely treatment, leading to irreversible retinal damage.

[0006] Therefore, there is a pressing clinical need for a reliable and non-invasive diagnostic technique to detect or predict retinopathy of prematurity (ROP) using measurable biomarkers. Traditional diagnostic methods, such as direct retinal tissue sampling, are highly invasive and risky, potentially leading to permanent retinal damage. Current technologies cannot accurately predict the likelihood of ROP in high-risk preterm infants. Therefore, this invention provides a biomarker-based non-invasive approach to address this clinical need, enabling early detection or prediction of ROP, thus facilitating timely clinical treatment and reducing the risk of blindness. Summary of the Invention

[0007] The purpose of this invention is to provide a method and system for solving the above-mentioned technical problems.

[0008] According to a first aspect of the present invention, a method for detecting the expression levels of proteomes related to retinopathy of prematurity in preterm infants is provided. The method includes: obtaining a tear sample from the preterm infant; extracting a protein expression profile from the tear sample; and detecting a proteome related to retinopathy of prematurity in the protein expression profile, which includes one or more upregulated and downregulated proteins.

[0009] The method further includes: calculating the log2 fold change (Log2FC) of each upregulated protein relative to its baseline expression level, and similarly calculating the log2 fold change of each downregulated protein; and generating a list including upregulated proteins with a log2 fold change greater than or equal to a positive threshold and downregulated proteins with a log2 fold change less than or equal to a negative threshold.

[0010] According to one embodiment of the present invention, the upregulated protein includes one or more of the following: β2 microglobulin (B2M), fatty acid-binding protein 5 (FABP5), protein S100-A4 (S100A4), secretory globulin family 1D member 1 (SCGB1D1), interleukin-1 receptor antagonist (IL1RN), glutathione reductase (GSR), GTP-binding nucleoprotein Ran (RAN), eukaryotic initiation factor 4A-I (EIF4A1), polymeric immunoglobulin receptor (PIGR), NAD(P)H dehydrogenase 1 (NQO1), cysteine-rich protein 1 (CRIP1), membrane-associated phospholipase A2 (PLA2G2A), DNA dC→dU editing enzyme APOBEC-3A (APOBEC3A), chondroitin acidic protein 1 (CRTAC1), and CXC motif chemokine 17 (CXCL17).

[0011] According to one embodiment of the present invention, the downregulated proteins include one or more of the following: antithrombin III (SERPINC1), fructose-1,6-bisphosphatase 1 (FBP1), apolipoprotein D (APOD), apolipoprotein AI (APOA1), apolipoprotein A-IV (APOA4), adenosine monophosphate kinase isoenzyme 1 (AK1), β-hexosinosinase α subunit (HEXA), hemoglobin-binding protein (HPX), alpha-fetoprotein (AFP), serum transferrin (TF), AMBP protein (AMBP), interproteasesin inhibitor heavy chain H2 (ITIH2), transthyretin (TTR), retinol-binding protein 4 (RBP4), apolipoprotein H (APOH), α-2-HS-glycoprotein (AHSG), and ganglioside GM2 activator protein (GM2A).

[0012] According to one embodiment of the present invention, the positive threshold is 0.58.

[0013] According to one embodiment of the present invention, the negative threshold is -0.58.

[0014] According to one embodiment of the present invention, the baseline performance is obtained by detecting tear samples from healthy premature infants.

[0015] According to one embodiment of the present invention, the protein expression spectrum is acquired by a data independent acquisition (DIA) mass spectrometer and quantified by a machine learning-based analysis process.

[0016] According to a second aspect of the present invention, a system for detecting the expression levels of a proteome associated with retinopathy of prematurity (ROP) in preterm infants is provided, comprising: a sample collection module configured to collect tear samples from preterm infants; a protein analysis module configured to extract a protein expression profile from the tear samples; and a protein detection module configured to detect the expression levels of a proteome associated with ROP in the protein expression profile, wherein the proteome comprises one or more upregulated proteins and one or more downregulated proteins.

[0017] The system further includes a calculation module configured to: calculate the log-2-fold change of each upregulated protein relative to a baseline expression level; calculate the log-2-fold change of each downregulated protein relative to a baseline expression level; and generate a list including upregulated proteins with a log-2-fold change greater than or equal to a positive threshold and downregulated proteins with a log-2-fold change less than or equal to a negative threshold.

[0018] According to one embodiment of the present invention, the upregulated protein includes one or more of the following: β2 microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, DNA dC→dU editing enzyme APOBEC-3A, chondroitin 1, and CXC motif chemokine 17. The downregulated proteins include one or more of the following: antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosinosinase α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activating protein.

[0019] According to one embodiment of the present invention, the baseline performance is obtained by detecting tear samples from healthy premature infants.

[0020] According to one embodiment of the present invention, the positive threshold is 0.58 and the negative threshold is -0.58.

[0021] According to one embodiment of the present invention, the protein analysis module includes a data-independent acquisition mass spectrometer.

[0022] According to a third aspect of the present invention, a kit is provided for detecting retinopathy of prematurity-related proteomes in preterm infants, comprising a protein assay for detecting the presence or expression levels of retinopathy of prematurity-related proteomes in preterm infant biological samples. The retinopathy of prematurity-related proteomes include one or more of the following: β2-microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, and DNA. dC→dU editing enzyme APOBEC-3A, chondroitin 1, CXC motif chemokine 17, antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosamine succinate α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, ganglioside GM2 activating protein.

[0023] According to one embodiment of the present invention, the protein detection method includes mass spectrometry detection, antibody immunoassay, protein chip or aptamer detection system.

[0024] According to one embodiment of the present invention, the biological sample includes tear fluid, blood, serum, or plasma samples. Attached Figure Description

[0025] Referring to the accompanying drawings, embodiments of the present invention will be described in more detail below, wherein:

[0026] Figure 1 A flowchart of a method for detecting the expression levels of proteomes associated with retinopathy of prematurity according to an embodiment of the present invention is shown.

[0027] Figure 2 A schematic block diagram of a system for detecting proteomic expression levels related to retinopathy of prematurity according to an embodiment of the present invention is shown.

[0028] Figures 3A-3D The characteristics of tear fluid samples are shown, among which Figure 3A Display gender ratio; Figure 3B This displays statistical values ​​showing the corrected age of premature infants; Figure 3C Display weight; and Figure 3D A histogram showing the severity of retinopathy of prematurity in preterm infants as assessed by an ophthalmologist, based on the region and stage of the disease.

[0029] Figures 4A-4BThe diagram shows tear protein volcanoes quantified using two analytical procedures. Figure 4A For using DIA-NN software in database-less mode; and Figure 4B PeakView software with a spectral database;

[0030] Figure 5 This section showcases proteins that exhibit differences in expression levels in both the analysis results of DIA-NN and PeakView software.

[0031] Figure 6 This demonstrates proteins that show differences in expression levels in both database-free DIA-NN software quantification and Spectronaut software analysis results;

[0032] Figure 7A and Figure 7B The presentation features rich visualization capabilities, including Figure 7A Enrichment for biological processes; Figure 7B For molecular functional enrichment; and

[0033] Figure 8 The receiver operating characteristic (ROC) analysis of the retrospective analysis of the protein SERPINC1 is shown, with an area under the curve of 0.758. Detailed Implementation

[0034] In the following description, methods and systems for detecting retinopathy of prematurity-related proteins are listed as preferred embodiments. It will be apparent to those skilled in the art that modifications, including additions and / or substitutions, can be made without departing from the scope and spirit of the invention. Specific details may be omitted below to avoid obscuring the invention; however, this document is prepared to enable those skilled in the art to practice the techniques disclosed herein without excessive experimentation.

[0035] Retinopathy of prematurity (ROP) can develop before immature retinal vessels extend to the retinal margin. Its severity is typically classified into five stages. Stage 1 is characterized by a white demarcation line at the junction of the vascularized and avascularized retina. Stage 2 involves the formation of a prominent, wide, and visible ridge at the junction of the vascularized and peripheral retina. Stage 3 is marked by abnormal angiogenesis at the ridge, with vascular growth and proliferation also observed within the ridge. Stage 4 involves partial retinal detachment, potentially requiring vitrectomy. Stage 5 involves complete retinal detachment, with poor treatment outcomes, ultimately leading to permanent vision loss. Furthermore, the location of ROP lesions can be divided into three zones based on the optic disc: Zone I, a small circular area centered on the optic disc with a radius twice the distance from the macula to the center of the optic disc; Zone II, an outer ring surrounding Zone I extending towards the nasal retinal margin; and Zone III, a crescent-shaped area located in the temporal retina.

[0036] According to a first aspect of the present invention, a method is provided for detecting the expression levels of proteomes related to retinopathy of prematurity in preterm infants. For example... Figure 1 As shown, the method first includes obtaining tear samples from the preterm infants in the study (step S101), wherein minimally invasive techniques can be used to collect tears, such as using Schirmer test strips or other ophthalmic sampling devices to collect a sufficient amount of tear samples from the surface of the eye.

[0037] After obtaining the sample, the tear sample is processed to obtain a complete protein expression profile (step S102). In some embodiments, the processing of the tear sample includes steps such as protein separation, denaturation, enzymatic hydrolysis, and further preparation of peptide fragments suitable for mass spectrometry analysis. Subsequently, high-throughput proteomics analysis of the protein expression profile is performed to detect and quantify the expression level of the target protein (step S103). In a preferred embodiment, the protein expression profile is acquired using a data-independent acquisition mass spectrometer to provide high accuracy, high reproducibility, and high coverage for tear protein quantification; furthermore, the obtained data can be further processed using machine learning-based analysis methods (e.g., neural network-driven algorithms) to improve detection accuracy and analyze its complex expression patterns.

[0038] In one embodiment, the method specifically focuses on detecting the expression levels of a predefined proteome associated with retinopathy of prematurity (ROP). This proteome includes upregulated and downregulated proteins associated with the pathophysiology of ROP. The upregulated proteins include one or more of the following: β2-microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, DNA dC→dU editing enzyme APOBEC-3A, chondroitin 1, and CXC motif chemokine 17.

[0039] On the other hand, the downregulated proteins include one or more of the following: antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosinosinase α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activating protein.

[0040] To assess the degree of difference in the expression levels of the aforementioned proteins in tear samples from preterm infants, the method further calculates the log-2 fold change value for each protein (step S104). More specifically, for upregulated proteins in the proteome, the expression level measured in the test preterm infant tear samples is compared with its corresponding baseline expression level to obtain the log-2 fold change value for upregulation; for downregulated proteins, the log-2 fold change value for downregulation is calculated using the same method. Preferably, the baseline expression level is obtained by calculating the expression level dataset of the corresponding proteins detected in tear samples from healthy preterm infants who have not been diagnosed with retinopathy of prematurity.

[0041] After calculating the log-2 fold change, the method further generates a list of proteins that meet the threshold criteria (step S105). More specifically, upregulated proteins with a log-2 fold change greater than or equal to a preset positive threshold (preferably 0.58) are included in the list; and downregulated proteins with a log-2 fold change less than or equal to a preset negative threshold (preferably -0.58) are also included in the list. This protein list can serve as a set of tear film molecular markers associated with retinopathy of prematurity, facilitating subsequent clinical decision-making or risk stratification assessment.

[0042] According to an embodiment of the present invention, a robust, minimally invasive, and data-driven approach can be provided to detect potential biomarkers for the risk of retinopathy of preterm birth (ROP) in preterm infants. Through quantitative proteomics and machine learning-based analysis, the present invention can identify molecular changes associated with ROP at an early stage, thereby enabling timely diagnosis and clinical intervention.

[0043] According to a second aspect of the present invention, a system is provided for detecting the expression levels of proteomes associated with retinopathy of prematurity in infants. For example... Figure 2 As shown, the system 200 can perform multi-stage operations. First, it includes a sample collection module 201 that can receive tear samples obtained from premature infants. The sample collection module 201 may include sampling tools, such as Schirmer test strips or microcapillary sampling devices, to obtain tear samples from the ocular surface in a minimally invasive manner.

[0044] System 200 further includes a protein analysis module 202. After tear sample collection is complete, the sample is transferred to the protein analysis module 202, from which protein expression profiles are extracted. The protein analysis module 202 can perform protein stabilization, cleavage, reduction, alkylation, enzymatic digestion (e.g., trypsin digestion), and subsequent peptide purification steps. In a preferred configuration, the protein analysis module 202 includes a data-independent acquisition mass spectrometer for high-throughput and reproducible tear protein quantification. This data-independent acquisition process can be combined with neural network-based computational processes or other machine learning-driven algorithms to support spectral deconvolution, protein identification, and quantification.

[0045] System 200 further includes a protein detection module 203. After protein analysis is completed, the sample is transferred to the protein detection module 203 to detect the expression levels of a set of proteomes associated with retinopathy of prematurity in the protein expression profile. This proteome includes one or more upregulated proteins and one or more downregulated proteins.

[0046] In one embodiment, the upregulated protein comprises one or more of the following: β2 microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, DNA dC→dU editing enzyme APOBEC-3A, chondroitin acidic protein 1, and CXC motif chemokine 17.

[0047] On the other hand, the downregulated proteins include one or more of the following: antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosinosinase α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activating protein.

[0048] In addition, system 200 includes a computation module 204 for performing multiple computational steps. These computational steps include calculating the log-2-fold change in the expression level of each upregulated protein relative to its corresponding baseline expression level; and calculating the log-2-fold change in the expression level of each downregulated protein relative to its corresponding baseline expression level. The baseline expression levels of the upregulated and downregulated proteins are derived from tear samples collected from healthy preterm infants who do not exhibit symptoms of retinopathy of prematurity. This serves as a reference dataset, providing a control benchmark to identify significant differences in the expression levels of proteins associated with disease development.

[0049] After calculating the log-2 fold change, the calculation module 204 of system 200 further generates a list, which includes: (i) upregulated proteins with a log-2 fold change greater than or equal to a preset positive threshold, and (ii) downregulated proteins with a log-2 fold change less than or equal to a preset negative threshold. In one embodiment, the positive threshold is 0.58 and the negative threshold is -0.58, which are commonly used and statistically validated fold change cutoff values ​​in protein somatic differential expression analysis.

[0050] In summary, this system 200 provides an integrated platform combining biosampling, mass spectrometry analysis, and computational analysis to detect and quantify biomarkers associated with retinopathy of prematurity (ROP) in the tears of preterm infants. Its output, a list of ROP-related proteins showing significant changes, is beneficial for guiding diagnosis, prognosis, and treatment in neonatal clinical care.

[0051] According to a third aspect of the present invention, a kit is provided for detecting a proteome associated with retinopathy of prematurity (ROP) in preterm infants. The kit includes a protein assay for detecting the presence or expression levels of ROP-associated proteins in a preterm infant biological sample. The proteome includes one or more of the following: β2-microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, and DNA. The following proteins are listed: dC→dU editing enzyme APOBEC-3A, chondroitin 1, CXC motif chemokine 17, antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosamine succinate α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteaseskin inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activator. These proteins, whether used alone or in combination, can serve as biomarkers for detecting disease states or risks, and have proven reliable and reproducible in clinical applications.

[0052] The term “protein detection method” as used in this article is broadly defined to encompass any detection technique that can identify, quantify or characterize a target protein. Therefore, it is not limited to a single analytical method, but includes all techniques that can reliably detect or measure the expression levels of one or more proteins associated with retinopathy of prematurity.

[0053] In some embodiments, the protein assays in this kit may include mass spectrometry-based assays. For example, liquid chromatography-tandem mass spectrometry (LC-MS / MS) for highly sensitive quantification of target peptides; independently acquired proteomics analysis for comprehensive analysis of complex samples; multiple reaction monitoring (MRM) for precise quantification of specific target proteins; and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) for rapid and high-throughput protein identification. Depending on laboratory equipment and specific needs, a suitable mass spectrometry technique can be selected for protein detection.

[0054] In some embodiments, the protein detection method may include antibody-based immunoassays. Suitable immunoassays include enzyme-linked immunosorbent assay (ELISA) for high-throughput quantitative detection in microplates; Western blotting to confirm the presence and molecular weight of proteins through separation and immunoassay; and Luminex bead multiplex immunoassay to simultaneously measure multiple retinopathy of prematurity-related proteins in a single sample. This antibody-based detection method helps improve compatibility with clinical and research laboratories.

[0055] In another embodiment, the protein detection method may include a protein chip in which immobilized antibodies or aptamers specifically bind to proteins in the proteome associated with retinopathy of prematurity. Such chips enable high-throughput screening of multiple proteins in limited samples, making them particularly suitable for precious or low-volume clinical samples such as preterm infant tears.

[0056] In some embodiments, the protein detection method may include an aptamer-based detection system. Aptamers are short nucleic acid or peptide molecules with high binding specificity, designed to specifically bind to one or more proteins associated with retinopathy of prematurity. Integrating an aptamer detection system into a kit provides a stable, reproducible alternative detection method suitable for both traditional laboratories and real-time diagnostic platforms.

[0057] This kit can detect biological samples including any clinically obtained bodily fluids from preterm infants. Applicable samples include tears, blood, serum, and plasma. This flexibility in sample selection enhances the kit's applicability to various clinical procedures and care settings while also addressing the need for low-invasiveness in preterm infant patients.

[0058] In summary, the kit of this invention provides a multifunctional and complete platform for detecting the expression of proteins associated with retinopathy of prematurity (ROP) in preterm infants. It can also incorporate multiple detection modalities, including mass spectrometry, antibody immunoassay, protein chips, and aptamer detection systems, to ensure adaptability to different laboratory environments and diagnostic needs. Furthermore, by analyzing diverse biological samples such as tears or blood, the kit of this invention combines clinical applicability with minimally invasive features, making it suitable for the more vulnerable preterm infant population.

[0059] Example

[0060] Definition of premature infant

[0061] Infants with a birth weight ≤ 1500 g or a gestational age ≤ 30 weeks were recruited from the Neonatal Intensive Care Unit of Queen Mary Hospital in Hong Kong as subjects. The trial was conducted by qualified pediatric ophthalmologists / retina specialists when the infants reached 4 weeks of age or 31 weeks of corrected age (whichever is later).

[0062] In a cross-sectional study of retinopathy of prematurity (ROP), tear samples were collected from both eyes of premature infants in the ROP and non-ROP groups using Schirmer test strips before each mydriatic instillation. During ROP screening, clinical diagnosis was performed according to the latest edition of the International Classification of Retinopathy of Prematurity (3rd Edition).

[0063] Example 1. Establishment of the retinopathy of prematurity proteome

[0064] To detect differences in protein expression associated with retinopathy of prematurity (ROP) in preterm infants, tear samples were collected from the subjects. Sampling was performed using Schirmer test strips. The strip was bent at a 90-degree angle at the cut end, and the subject's lower eyelid was gently pulled. The bent end of the strip was placed at the junction of the lower eyelid and the strip, absorbing at least 20 mm of the marked area. The test strips were then immediately subjected to dry heat treatment to preserve the collected proteins, and transported in microcentrifuge tubes at room temperature to the laboratory for proteomics analysis.

[0065] Tear samples were collected by qualified ophthalmologists at Queen Mary Hospital, Hong Kong. A total of 33 tear samples were collected from 34 premature infants, of whom 4 were diagnosed with retinopathy of prematurity (ROP) and 19 were not. Samples were collected from both eyes on the day of the clinical diagnosis, resulting in 13 ROP-related samples and 21 non-ROP samples. The study population included 20 males and 14 females (e.g., ...). Figure 3A (As shown). Statistical analysis results showed that the corrected age of the retinopathy of preterm infant group and the non-retinopathy of preterm infant group was 37.4 ± 2.9 weeks vs. 35.8 ± 2.0 weeks, p = 0.103; Figure 3B ) and weight (37.4 ± 2.9 g vs. 35.8 ± 2.0 g, p = 0.297; Figure 3C There was no significant difference between the two groups. Most cases of retinopathy of prematurity diagnosed belong to stage II in zone II. Figure 3D Statistical comparisons were performed using an unpaired Student's t-test with unequal variances.

[0066] The total protein concentration in the collected tear samples ranged from 3.5 to 9.5 mg / mL in both basal and reflective tears, with higher concentrations (11-13 mg / mL) observed in neonatal tears. During protein extraction, each Schirmer test strip was cut into approximately 2 mm segments and placed in new centrifuge tubes. After elution with 5% SDS buffer and shaking at room temperature for 1 hour, the extract was centrifuged to separate the extract. Protein concentration was then determined using the biuret acid (BCA) protein quantification method. The extracted proteins were reduced, alkylated, enzymatically digested, purified, and reconstituted in 0.1% formic acid solution. The final peptide concentration was adjusted to 0.5 μg / μL for subsequent mass spectrometry analysis. Data acquisition was performed using continuous window full theoretical mass spectrometry (SWATH-MS), and the obtained data were analyzed using DIA-NN software in database-free mode and PeakView software. Protein identification and quantification were performed by matching a computer-generated trypsin digestion peptide profile from the UniProt human protein database with neural network dynamics analysis.

[0067] Using DIA-NN software, 332 proteins were quantified at a 1% false discovery rate (FDR). Among these, 53 proteins showed statistically significant differences in expression levels between preterm and non-preterm retinopathy of prematurity (RRP) groups, defined as a logarithmic change ≥ 0.58 or ≤ -0.58, with p < 0.05. Figure 4A (As shown). Simultaneously, PeakView software was used for analysis, and a spectral database based on human tears was established (obtained from independent data acquisition mass spectrometry of mixed samples). This method quantified a total of 417 proteins (1% false detection rate), of which 73 proteins showed significant differences (…). Figure 4B ).

[0068] Further comparison of the DIA-NN and PeakView analysis workflows confirmed that 15 proteins showed significant differences between the preterm retinopathy of prematurity (DRP) and non-DRP groups on both platforms (p < 0.05, false discovery rate 1%). These differences are expressed as log-2 changes, shown as mean ± standard deviation (SD).

[0069] The proteins upregulated in the retinopathy of prematurity group included: β2-microglobulin (1.59 ± 0.04), fatty acid-binding protein 5 (1.19 ± 0.37), protein S100-A4 (1.26 ± 0.10), secretory globulin family 1D member 1 (1.36 ± 0.12), interleukin-1 receptor antagonist (1.06 ± 0.28), glutathione reductase (0.82 ± 0), GTP-binding nucleoprotein Ran (0.65 ± 0.02), and eukaryotic initiation factor 4A-I (1.13 ± 0.76).

[0070] Proteins downregulated in the retinopathy of preterm infant group included: antithrombin III (-0.91 ± 0.35), fructose-1,6-bisphosphatase 1 (-0.92 ± 0.14), apolipoprotein D (-1.00 ± 0.05), apolipoprotein AI (-1.39 ± 0.60), apolipoprotein A-IV (-1.51 ± 0.40), and adenosine monophosphate kinase isoenzyme 1 (-1.44 ± 0.27). Figure 5 (As shown).

[0071] To further identify proteins in tears associated with retinopathy of prematurity, this embodiment employed the Direct Data Independent Acquisition (DIA+) mode of Spectronaut software for analysis, and used a human proteome containing 20,434 reviewed items for protein identification. Standardized BGS factory settings (preset) were used for analysis parameters; the proteolytic enzyme was set to trypsin / P, allowing a maximum of two missed cleavage sites; fixed modification was cysteine ​​carboxymethylation; variable modifications included N-terminal acetylation and methionine residue oxidation; peptide length was limited to 7 to 52 amino acids; and a 1% false detection rate was set at both the peptide precursor and protein levels to ensure high-confidence identification. Protein quantification was performed using the MaxLFQ algorithm with automatic normalization enabled.

[0072] Parallel analysis was performed using the database-free mode of DIA-NN software, with a predicted spectral database generated from the same FASTA file for independent data acquisition, searching, and quantification. The digestive enzyme was also set to trypsin / P; a maximum of two missed cleavage sites were allowed; modification conditions included cysteine ​​carboxymethylation, N-terminal methionine cleavage, oxidation (M), and acetylation (N-terminus); peptide length was limited to 7 to 52 amino acids; precursor ion charge range was limited to 2 to 5; and both mass precision and MS1 precision were set to automatic determination. Cross-sample matching (match between runs) was employed to promote comparability between samples, and retention time (RT)-dependent cross-experimental normalization and a "robust LC (high precision)" quantification strategy were used to improve data consistency.

[0073] Based on this proteomics analysis method, a total of 18 proteins were found to show significant differences between the retinopathy of preterm (ROP) group and the non-ROP group (false discovery rate 1%, p < 0.05). Proteins showing upregulated expression in the ROP group included: polymeric immunoglobulin receptor (1.25±0.35), NAD(P)H dehydrogenase 1 (1.2±0.28), cysteine-rich protein 1 (1.1±0.14), membrane-associated phospholipase A2 (1.2±0), NA dC→dU editing enzyme APOBEC-3A (0.85±0.07), chondroitin 1 (0.8±0.14), and CXC motif chemokine 17 (1.05±0.21). Proteins with downregulated expression levels in the retinopathy of preterm infant group included: β-hexosinosinase α subunit (-0.7 ± 0.14), hemoglobin-binding protein (-0.8 ± 0.14), alpha-fetoprotein (-0.9 ± 0.14), serum transferrin (-0.8 ± 0), AMBP protein (-1.1 ± 0.14), interproteases inhibitor heavy chain H2 (-1 ± 0.14), transthyretin (-1.1 ± 0.42), retinol-binding protein 4 (-1.3 ± 0.14), apolipoprotein H (-1.15 ± 0.35), α-2-HS-glycoprotein (-1.5 ± 0.14), and ganglioside GM2 activator protein (-2.15 ± 0.64). Figure 6 (As shown). These proteins are involved in pathways related to angiogenesis, cell survival, angiogenesis, and metabolic dysregulation, suggesting their potential relevance in mediating the response to hypoxia and oxidative stress in the pathophysiology of retinopathy of preterm infants.

[0074] Example 2. Protein functional enrichment and biomarker analysis applied to the screening of retinopathy of prematurity.

[0075] Using the integrated Gene Ontology (GO) annotation tool on the STRING software platform, 33 proteins with differential expression levels were analyzed to determine whether overexpressed biological processes and molecular functions existed. The analysis revealed significant enrichment of biological processes and molecular functional pathways related to lipid metabolism and antioxidant activity. Both pathways play crucial roles in angiogenesis and oxidative stress response, and these processes are also pathologically dysregulated in retinopathy of preterm birth. Therefore, the enrichment results provide further important insights into the potential driving mechanisms of disease progression (e.g., ...). Figure 7A and Figure 7B (As shown).

[0076] To evaluate the clinical applicability of the proteomics provided by this invention, receiver operating characteristic (ROC) curve analysis was further used to analyze single protein biomarkers. Antithrombin III was used as the analysis target, showing consistent differential expression across multiple analyses. The area under the ROC curve (AUC) of the ROC curve analysis results was 0.758, indicating statistically significant discriminatory ability in distinguishing between retinopathy of preterm and non-preterm infants. Retrospective fitting results based on antithrombin III protein expression data showed a sensitivity of 81% and a specificity of 62% (e.g., ...). Figure 8 (As shown in the figure). The above results demonstrate the diagnostic potential of individual protein biomarkers (such as antithrombin III) in non-invasive tear testing and lay the foundation for the future development of multiple biomarker groups to improve diagnostic efficacy and clinical applicability, thereby enhancing diagnostic performance and clinical suitability.

[0077] As used herein, the terms “approximately,” “substantially,” “essentially,” and “about / approximately” are used to describe and explain a small variation. When used in conjunction with an event or situation, the term can refer to the exact occurrence of the event or situation, or approximately the occurrence of the event or situation. The term “about,” as used herein with respect to a given value or range, typically refers to a range of ±10%, ±5%, ±1%, or ±0.5% of the given value or interval, which can be understood herein as ranging from one endpoint to another or between two endpoints. Unless otherwise stated, all ranges disclosed in this disclosure include endpoints. When referring to the same numerical value or characteristic, the term may refer to a value within ±10%, ±5%, ±1%, or ±0.5% of the average of the values.

[0078] The systems and methods disclosed in the embodiments of the present invention can be implemented by computing devices, computer processors, or electronic circuits, including but not limited to application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), central processing units (CPUs), graphics processing units (GPUs), and other programmable logic devices configured or programmed according to the teachings of the present invention. Computer instructions or software code running on computing devices or programmable logic devices can be implemented by those skilled in the art based on the disclosures of the present invention.

[0079] The methods or some steps of the present invention can be executed on one or more computing devices, which may include server computers, personal computers, laptops and mobile computing devices such as smartphones and tablets.

[0080] The functional units and modules according to embodiments of the present invention can also be implemented in a distributed computing environment and / or a cloud computing environment; and all or part of the machine instructions can be executed in a distributed manner by one or more processing devices through a communication network route such as an intranet, a wide area network (WAN), a local area network (LAN), the Internet and other forms of data transmission media.

[0081] The above description is provided for the purpose of illustrating and describing the invention, and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0082] The above embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling other skilled in the art to understand the various embodiments of the invention and the various modifications suitable for the intended particular use.

Claims

1. A method for diagnosing retinopathy of prematurity (ROP) in preterm infants by detecting and calculating the expression levels of proteomes associated with ROP, characterized in that, include: Tear samples were obtained from the premature infants. Protein expression profiles were extracted from the tear fluid samples; The retinal disease-related proteome, including one or more upregulated and downregulated proteins, was detected in the protein expression profile. Calculate the log-2 change of each upregulated protein relative to its baseline expression level, and similarly calculate the log-2 change of each downregulated protein. as well as A list of proteins is generated as molecular characteristics of tear biomarkers related to retinopathy, wherein the protein list includes: The upregulated proteins whose log-2 change value is greater than or equal to a positive threshold; and The downregulated proteins whose log-two change value is less than or equal to the negative threshold; The upregulated proteins include one or more of the following: β2-microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, and DNA. The dC→dU editing enzyme APOBEC-3A, chondroitin 1, CXC motif chemokine 17; and the downregulated proteins include one or more of the following: antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase 1, β-hexosinosinase α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activating protein.

2. The method according to claim 1, wherein the positive threshold is 0.

58.

3. The method according to claim 1, wherein the negative threshold is -0.

58.

4. The method of claim 1, wherein the baseline expression level is obtained by detecting relative proteins in tear samples from healthy preterm infants.

5. The method according to claim 1, wherein the protein expression spectrum is acquired by a data-independent acquisition mass spectrometer and quantified by a machine learning-based analysis step.

6. A system for diagnosing retinopathy of prematurity (ROP) in preterm infants by detecting and calculating the expression levels of proteomes associated with ROP, characterized in that, include: The sample collection module is configured to collect tear samples from premature infants. A protein analysis module is configured to extract a protein expression profile from the tear sample. A protein detection module is configured to detect the expression level of a set of proteomes associated with retinopathy of prematurity in the protein expression profile, wherein the proteomes include one or more upregulated proteins and one or more downregulated proteins. as well as The calculation module is configured as follows: Calculate the log-two change in the expression level of each upregulated protein relative to the baseline expression level; Calculate the log-two change in the expression level of each downregulated protein relative to the baseline; as well as A protein list will be generated as a molecular feature of tear biomarkers associated with retinopathy to facilitate subsequent clinical decision-making or risk stratification. This protein list will include: The upregulated proteins whose log-two change value is greater than or equal to the positive threshold; as well as The downregulated proteins are those whose logarithmic double change value is less than or equal to the negative threshold.

7. The system of claim 6, wherein the upregulated protein comprises: One or more of the following: β2 microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, DNA dC→dU editing enzyme APOBEC-3A, chondroitin 1, and CXC motif chemokine 17.

8. The system of claim 6, wherein the downregulated protein comprises: One or more of the following: antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosinosinase α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, and ganglioside GM2 activating protein.

9. The system of claim 6, wherein the baseline performance level is obtained by detecting relative protein levels in tear samples from healthy preterm infants.

10. The system of claim 6, wherein the positive threshold is 0.58 and the negative threshold is -0.

58.

11. The system of claim 6, wherein the protein analysis module includes a data-independent acquisition mass spectrometer.

12. A kit for detecting proteomes associated with retinopathy of prematurity in preterm infants, characterized in that, include: A protein assay used to detect the presence or expression level of the proteome associated with retinopathy of prematurity in biological samples of preterm infants. The proteome associated with retinopathy of prematurity (ROP) in these preterm infants includes: β2-microglobulin, fatty acid-binding protein 5, protein S100-A4, secretory globulin family 1D member 1, interleukin-1 receptor antagonist, glutathione reductase, GTP-binding nucleoprotein Ran, eukaryotic initiation factor 4A-I, polymeric immunoglobulin receptor, NAD(P)H dehydrogenase 1, cysteine-rich protein 1, membrane-associated phospholipase A2, and DNA. dC→dU editing enzyme APOBEC-3A, chondroitin 1, CXC motif chemokine 17, antithrombin III, fructose-1,6-bisphosphatase 1, apolipoprotein D, apolipoprotein AI, apolipoprotein A-IV, adenosine monophosphate kinase isoenzyme 1, β-hexosamine succinate α subunit, hemoglobin-binding protein, alpha-fetoprotein, serum transferrin, AMBP protein, interproteases inhibitor heavy chain H2, transthyretin, retinol-binding protein 4, apolipoprotein H, α-2-HS-glycoprotein, ganglioside GM2 activating protein.

13. The kit according to claim 12, wherein the protein detection method includes mass spectrometry, antibody immunoassay, protein chip or aptamer detection system.

14. The kit of claim 12, wherein the biological sample comprises tears, blood, serum or plasma.

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