Liver ischemia reperfusion injury biomarker based on proteomics and application thereof

Abstract

The invention discloses a biomarker for hepatic ischemia reperfusion injury based on proteomics, the biomarker is NGP (Neutrophil Granule Protein), and the biomarker can be used for early diagnosis and prevention, prognosis monitoring and judgment and the like of complications after hepatic resection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a proteomics-based biomarker for liver ischemia-reperfusion injury and its application. Background Art

[0002] Despite advances in surgical techniques and perioperative care, the incidence of complications after liver resection remains high, especially in patients with cirrhosis, who often have concurrent liver function impairment, coagulopathy, and reduced liver reserve, leading to a decreased tolerance for surgery. Therefore, surgeons urgently need to reduce the risk of liver resection by using various preoperative assessments to stratify risk and determine the feasibility and extent of liver resection.

[0003] Currently, the identification of useful biomarkers for predicting the risk of postoperative complications after liver resection remains insufficient, and available serum biomarkers show low sensitivity and heterogeneous specificity. There is an urgent need to find one or more available serum biomarkers with high sensitivity and specificity to predict perioperative complications and mortality after liver resection, so as to provide a basis for clinical or medical researchers to determine whether liver resection is feasible and the extent of liver resection.

[0004] Proteomics is a science that uses proteins as research objects to study the protein composition and activity patterns of cells, tissues, or entire organisms at a holistic level. Proteomics studies protein characteristics, including protein expression levels, post-translational modifications, and protein-protein interactions, thereby gaining a comprehensive understanding of disease occurrence and progression at the protein level. In proteomics, the key technology for protein identification in biological samples is mass spectrometry (MS). Hepatic ischemia-reperfusion injury (HIRI) is a major pathological mechanism for postoperative complications after hepatectomy. Because the mechanism of HIRI is a complex process involving numerous factors, the use of proteomics can help people gain a deeper understanding of the pathogenesis and treatment of HIRI.

[0005] Neutrophil granule protein (NGP) is a protein with a molecular weight of approximately 19.33 kDa, primarily found in the granules of neutrophils. While limited research has directly investigated the function of NGP, it is speculated that, based on the biological properties of neutrophils, NGP may be involved in antimicrobial defense and inflammation regulation. However, research on the role of NGP in hepatic ischemia-reperfusion injury remains preliminary, and its predictive value for postoperative complications in patients undergoing hepatectomy remains unclear. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a proteomics-based biomarker for liver ischemia-reperfusion injury and its application, which can be used for early diagnosis and prevention of complications after liver resection, prognosis monitoring and judgment, etc.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a proteomics-based biomarker for liver ischemia-reperfusion injury, wherein the marker is NGP. The present invention firstly detects the liver tissue proteomics of mice 6h and 24h after liver ischemia-reperfusion (IR) and finds that neutrophil granule protein (NGP) is significantly upregulated after IR. The serum NGP of patients after liver resection is further detected and it is found that NGP on the first day after surgery can be used as a marker for predicting complications after liver resection. Serum NGP levels can well predict the risk of complications after liver resection.

[0009] Furthermore, the biomarker is used to detect a sample by proteomics analysis, and the sample is from liver tissue.

[0010] The second aspect of the present invention provides the use of the neutrophil granule protein (NGP) in the preparation of a reagent, a kit, a microarray or a biochip for predicting postoperative complications in clinical liver resection patients.

[0011] Furthermore, the neutrophil granule protein is human serum neutrophil granule protein.

[0012] Furthermore, the clinical postoperative complications of liver resection patients are severe complications of grades III-V according to the Clavien-Dindo classification, such as bleeding, liver failure, abdominal infection, bile leakage, pleural effusion and pulmonary complications, ascites, etc.

[0013] Furthermore, the reagent or kit includes immunoglobulin G antibody against neutrophil granule protein.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides biomarkers for analyzing the risk of post-hepatectomy complications and their applications. Using these biomarkers, reagents or kits for analyzing the risk of post-hepatectomy complications can be prepared to predict the risk of clinical post-hepatectomy complications.

[0016] The biomarkers provided by the present invention will help to better understand the pathophysiology of clinical post-hepatectomy complications and will provide new opportunities for diagnosis and prognosis, thereby improving clinical services for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a box plot of the whole proteome data of Example 1 of the present invention.

[0018] Figure 2 This is the PCA diagram of the whole proteome of Example 1 of the present invention.

[0019] Figure 3 This is the DEPs volcano plot of Example 1 of the present invention, with red markings indicating upregulated proteins, gray markings indicating proteins with no differential changes, and blue markings indicating downregulated proteins.

[0020] Figure 4 This is the DEPs clustering heat map analysis of Example 1 of the present invention, blue represents up-regulated proteins, and turquoise represents down-regulated proteins.

[0021] Figure 5 This is a bubble diagram of the whole proteome differential protein G0 analysis in Example 1 of the present invention.

[0022] Figure 6 This is the whole protein level NGP Western Blot of Example 1 of the present invention.

[0023] Figure 7 This is a diagram showing the effect of Western Blot on the expression of NGP in liver tissue in the present invention.

[0024] Figure 8 This is a diagram showing the diagnostic effect of the mouse liver ischemia-reperfusion injury model of the present invention. DETAILED DESCRIPTION

[0025] Below in conjunction with specific embodiment, further elaborate on the design of the present invention, positive sample verification and result analysis.It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0026] Example 1

[0027] 1 Experimental materials and instruments

[0028] 1.1 Animal Source

[0029] All experiments used adult male SPF-grade C57BL / 6 mice aged 8–10 weeks and weighing 20–23 g (purchased from the Animal Center of Xi’an Jiaotong University).

[0030] 1.2 Main reagents for the experiment

[0031] BCA protein quantification kit (Thermo); Western and IP cell lysis buffer (Meilunbio); protease inhibitor cocktail (PD) (Roche); phosphatase inhibitor (PPI) (Roche); formic acid (FA) (mass spectrometry grade) (Sigma-Aldrich); trifluoroacetic acid (TFA) (Sigma-Aldrich); acetonitrile (ACN) (mass spectrometry grade) (Thermo Fisher Scientific); anhydrous methanol (Guangzhou Chemical Reagent Factory); iodoacetamide (IAA) (Sigma-Aldrich); dithiothreitol (DTT) (Sigma-Aldrich); urea (UREA) (Sigma-Aldrich); trypsin (Shengxia protein V5280); anhydrous ethanol (Guangzhou Chemical Reagent Factory); tetraethylammonium bromide (TEAB) (Sigma-Aldrich); iRT Kit (Biognosys); Tween (Sigma-Aldrich); ECL chemiluminescent substrate (Bio-Rad); PVDF membrane; 5× Loading Buffer (Bio-Rad); Protein Marker (Bio-Rad); Skim milk powder (Genebase); Ammonium persulfate (APS) (Sigma-Aldrich); Sodium dodecyl sulfate (SDS) (Sigma-Aldrich); Tetramethylethylenediamine (TEMED) (Sigma-Aldrich); Acrylamide (Sigma-Aldrich); 1.5 M Tris-HCl buffer (Sigma-Aldrich).

[0032] 1.3 Primary Antibodies

[0033] Rabbit anti-NGP monoclonal antibody (1:1000) (Proteintech); GAPDH polyclonal antibody (1:2000) (Proteintech); rabbit secondary antibody (1:5000) (Proteintech).

[0034] 1.4 Main experimental instruments

[0035] Ice maker (Henan Tianchi Instrument Equipment Co., Ltd.); electronic balance (Doris Scientific Instrument (Beijing) Co., Ltd.); low-temperature tissue grinder (Dinghaoyuan (Tianjin) Biotechnology Co., Ltd.); non-contact ultrasonic instrument (Ningbo Xinzhi Biotechnology Co., Ltd.); refrigerated centrifuge (Eppendorf, Germany); mass spectrometer (Thermo Fisher Scientific); microplate reader (Xi'an Saiya Technology Co., Ltd.); vacuum desalination pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.); ELGA PURELAB Classic UV water purifier (Guangzhou Shenhua Biotechnology Co., Ltd.); constant temperature incubator shaker (Shanghai Zhichu Instrument Co., Ltd.); water bath (Beijing Changfeng Instrument Co., Ltd.); vortex shaker (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.); freeze dryer (Hunan Hexi Instrument Equipment Co., Ltd.); protein gel electrophoresis apparatus (Xi'an Tengling Biotechnology Co., Ltd.); multi-color fluorescence and chemiluminescence gel imaging system (Times Legend Biotechnology Co., Ltd.).

[0036] 1.5 Preparation of main reagents

[0037] (1) Mass spectrometry experiment related reagent formula

[0038] a) 8M Urea: Dissolve 24.024 g of Urea in 50 mL of water and adjust the pH to 8.0 with concentrated hydrochloric acid.

[0039] b) 1M DTT: 154.2 mg DTT was dissolved in 1M gold water.

[0040] c) 1M IAA: 184.96 mg IAA was dissolved in 1 mL gold water.

[0041] d) Conditioning Buffer: 0.1% TFA dissolved in 20% acetonitrile solution.

[0042] e) Washing Buffer: 0.1% TFA dissolved in 5% acetonitrile solution.

[0043] f) Elution Buffer: 0.1% TFA dissolved in 60% acetonitrile solution.

[0044] (2) Western Blot related reagent formula

[0045] a) 5% stacking gel, ultrapure water 2.1 mL, 30% monomer 0.5 nL, 1.5 M Tris-HCl pH 6.8 0.38 mL, 10% SDS 30 μL, 10% APS 30 μL, TEMED 3 μL.

[0046] b) 10% separating gel, 6.9 mL of ultrapure water, 4 mL of 30% monomer, 0.75 mL of 1.5 M Tris-HCl pH 8.8, 60 μL of 10% SDS, 60 μL of 10% APS, and 9 μL of TEMED.

[0047] c) Electrophoresis buffer (10×), Tris-base 30.3 g, Glycine (glycolic acid) 144.4 g, SDS 10 g, pure water 1000 mL.

[0048] d) Transfer buffer (10×), Tris-base 37.9 g, Glycine 187.7 g, and pure water 1000 mL.

[0049] e) Transfer buffer 1×, 10× transfer buffer 80 mL, anhydrous methanol 200 mL, pure water 720 mL.

[0050] f) TBS buffer (10×), sodium chloride 80 g, potassium chloride 2 g, Tris-base 30 g, and pure water 1000 mL.

[0051] g) TBST buffer (1×), 50 mL of 10× TBS buffer, 500 μL of Tween 20, and 450 μL of pure water.

[0052] h) 5% skim milk, 2.5 g skim milk powder, and 50 L of 1× TBST buffer.

[0053] 2 Experimental methods

[0054] 2.1 Establishment of a mouse liver ischemia-reperfusion injury model

[0055] (1) In this example, a liver ischemia-reperfusion injury model was established in adult male SPF C57BL / 6 mice aged 8-10 weeks and weighing 20-23 g. Six mice were randomly divided into two groups: a sham group (Sham) and an injury group (Injury).

[0056] (2) The mice in the injury group were used as the experimental group. ① The mice were anesthetized by intraperitoneal injection of anesthetics (such as 3% isoflurane gas or sodium pentobarbital). ② A midline incision was made in the abdomen, the abdominal cavity was opened, and the hepatic pedicles of the left and middle lobes of the liver (including the portal vein and hepatic artery) were carefully separated. ③ The portal vein and hepatic artery of the middle and left lobes were clamped with non-invasive vascular clamps, causing about 70% liver ischemia. ④ After 0.5 minutes, compared with the non-blocked right lobe, the blocked lobe was significantly whitened by the naked eye, indicating that the blockage was successful. The skin incision was clamped with hemostatic forceps to temporarily close the abdominal cavity, and the mice were placed on a 37°C constant temperature heating pad to keep warm. During the interoperative period, a wet cotton pad was used to cover the incision to prevent fluid loss. ⑤ After 1 hour of continuous ischemia, the vascular clamp was quickly removed, and the liver tissue was observed to turn red, confirming blood recirculation. After the model was successfully established, the subcutaneous tissue and skin were sutured in turn. After reperfusion for 6 h and 24 h, the mice were deeply anesthetized with sodium pentobarbital again by intraperitoneal injection. The ischemic liver tissues of the mice were extracted, rinsed three times with PBS, placed in 2 mL cryovials, quickly frozen with liquid nitrogen, and stored in a refrigerator at -80°C.

[0057] (3) The mice in the sham-operated group served as the control group. Only the skin was incised without liver treatment, and the subcutaneous tissue and skin were sutured in sequence.

[0058] 2.3 Tissue grinding and lysis

[0059] (1) Take out the mouse liver tissue from the -80℃ refrigerator and place it on dry ice. Cut the liver tissue into pieces on the clean bench and put them into 2mL grinding tubes. Clean the grinding beads with anhydrous ethanol and dry them with absorbent paper. Add 100 small grinding beads to each grinding tube and grind them with a pre-cooled tissue grinder (working conditions: 70Hz, 2min, -50℃ grinding until powdered).

[0060] (2) Add 1 mL of cell lysis buffer containing protease inhibitors and phosphatase inhibitors to the tissue grinding tube. Remove the grinding beads from the tissue grinding tube with tweezers, precool to 4°C using non-contact ultrasonication, start the precooling system water circulation, and ultrasonically disrupt the tissue in the grinding tube (4°C, ultrasonication for 5 seconds with 10 seconds intervals, for 10 minutes). The tissue particles are completely lysed.

[0061] (3) Place the grinding tube containing the tissue on ice for 30 minutes, shaking vigorously every 10 minutes. The lysed tissue is centrifuged in a pre-cooled centrifuge (working conditions: 4°C, 12000 rpm, 30 minutes), and the supernatant is transferred to a 1.5 mL centrifuge tube using a pipette.

[0062] 2.4 High-efficiency protein enzymatic hydrolysis

[0063] (1) The liver protein sample extracted in the previous step was tested for protein concentration using a BCA kit: the protein standard was diluted with ultrapure water to prepare standard samples with final concentrations of 0 μg / μL, 0.0625 μg / μL, 0.125 μg / μL, 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL, and 2 μg / μL, respectively. The BCA working solution was prepared at a ratio of 200 μL of B solution and 4 μL of A solution per sample, incubated in a 37°C incubator for 30 minutes, and the absorbance at a wavelength of 570 nm was read using a microplate reader. According to the protein concentration detected by the BCA kit, 3 mg of the corresponding volume of protein sample was placed in a 15 mL centrifuge tube for enzymatic hydrolysis.

[0064] (2) Add an appropriate volume of 8M Urea to a 15 mL centrifuge tube to make the final concentration greater than 4M (i.e., 1:1). The sample with the lowest concentration shall be used as the standard. The remaining samples with higher concentrations shall be filled to the same volume with 8M Urea and shaken thoroughly.

[0065] (3) Prepare 1M DTT (prepared in gold water), add an appropriate volume to make its working concentration 50mM (i.e. 1:20), shake thoroughly, and incubate in a 37°C water bath for 1 hour to fully reduce the disulfide bonds of the protein.

[0066] (4) Prepare 1 M IAA (prepared with gold water), add an appropriate volume to make its working concentration 135 mM, shake thoroughly, and place at room temperature in the dark for 30 minutes.

[0067] (5) Rinse the ultrafiltration tube with 1 mL of TEAB and centrifuge (working conditions: 20°C, 4000 rpm, 20 min). If the liquid is not completely separated, change the centrifugal angle by 90° to avoid damage to the ultrafiltration tube membrane. Continue centrifuging until all the liquid is separated. Each centrifugation should not exceed 15 min to avoid damage to the ultrafiltration tube membrane.

[0068] (6) Add the sample to the rinsed ultrafiltration tube. The volume of each tube should not exceed 1 mL. Centrifuge (working conditions: 20°C, 4000 rpm, 20 min). If the liquid is not completely separated, change the centrifugal angle 90° to avoid damage to the ultrafiltration tube membrane. Centrifuge until all the liquid is separated. Each centrifugation should not exceed 15 min to avoid damage to the ultrafiltration tube membrane.

[0069] (7) Add 1 mL of 8 M Urea to the ultrafiltration tube, shake thoroughly, and centrifuge until all the liquid is removed (working conditions: 4°C, 4000 rpm, 20 min), and repeat twice.

[0070] (8) Add 1 mL of TEAB to the ultrafiltration tube, shake thoroughly, and centrifuge (working conditions: 4°C, 4000 rpm, 20 min). Centrifuge until all the liquid is removed. Repeat 5 times. Discard the collection tube and replace it with a new one.

[0071] (9) Dissolve pancreatic enzyme in the company's HCl solution to prepare a pancreatic enzyme concentration of 1 μg / μL. Add 500 μL of 50 mM TEAB and 75 μL of the prepared mass spectrometry-grade pancreatic enzyme solution to the ultrafiltration tube in sequence.

[0072] (10) Use a clean gun tip to blow away the flocculent protein in the ultrafiltration tube to make the solution turbid, shake it thoroughly for 5 minutes, seal the ultrafiltration tube with plastic wrap, place it in a shaker at 37°C and 200 rpm, and shake it for 16-18 hours. After 8 hours, use a clean gun tip to blow away the flocculent protein again to make the solution turbid, and shake it thoroughly for 5 minutes.

[0073] (11) Centrifuge the ultrafiltration tube from the previous step into a collection tube (working conditions: 4°C, 4000 rpm, 20 min), add 1 mL of gold water, use a clean gun tip to blow away the flocculent protein to make the solution turbid, shake it thoroughly for 5 min, and centrifuge it (working conditions: 4°C, 4000 rpm, 20 min) to collect the enzymatic peptide solution.

[0074] (12) After enzymatic hydrolysis, the peptide solution was tested for concentration using a BCA kit and the enzymatic hydrolysis efficiency of the protein sample was calculated.

[0075] 2.5 High-throughput desalination

[0076] (1) Connect the vacuum desalination instrument and check for tightness. Clean the desalination column connector with pure water.

[0077] (2) Activation of desalting column: Add methanol to activate Sep-pak C18 desalting column for 10 minutes. The vacuum pump pressure should not exceed 300 kPa.

[0078] (3) Equilibration of desalting column: Prepare 2% acetonitrile containing 0.1% FA as the equilibration buffer (Condition Buffer) and pass it through the column twice, 1 mL each time, and let it stay on the column for 1 min for the first pass.

[0079] (4) Absorb the sample: Place the waste liquid tube on the rack of the negative pressure tank, and pass the enzymatically hydrolyzed peptide solution through the column 3-5 times, 1 mL each time. The first pass should be allowed to stay on the column for 1 minute. Use a vacuum pump to remove salt. Remember not to make the pressure difference too large, which will cause the flow rate to be too fast and generate bubbles, thereby leaking.

[0080] (5) Sample cleaning: Prepare gold water containing 0.1% FA as washing buffer, 1 mL each time: repeat 5 times.

[0081] (6) Elution of samples: Place the collection tube on the rack of the negative pressure tank, first use 0.1% FA in 40% acetonitrile as the elution buffer (Elution Buffer), 200 μL each time, and pass through the column three times.

[0082] (7) Use 0.1% FA in 80% acetonitrile as the elution buffer again, 200 μL each time, and pass through the column three times. Take 200 μL of the peptide sample solution for whole protein spectrum detection, and freeze-dry the desalted peptide sample solution using a cold trap and a vacuum pump. The remaining sample is freeze-dried using a cold trap and a vacuum pump.

[0083] 2.7 Liquid chromatography coupled with mass spectrometry (LC-MS / MS)

[0084] (1) After desalting, the freeze-dried whole protein sample was dissolved in 20 μL of 0.1% FA (prepared with gold water) and the concentration was detected using a BCA kit. The amount of 0.1% FA to be added was adjusted according to the measured concentration to make the final concentration of the sample 0.5 μg / μL.

[0085] (2) Removal of particulate matter: Transfer 15 μL of each sample to a new EP tube and centrifuge (working conditions: 4°C, 12,000 g, 20 min). Transfer 12 μL of the supernatant to a new centrifuge tube and centrifuge (working conditions: 4°C, 12,000 g, 20 min). Transfer 9.5 μL of the supernatant to a new centrifuge tube, add 0.5 μL of the labeled peptide (Irt), vortex to mix, and centrifuge (working conditions: 4°C, 12,000 g, 10 min).

[0086] (3) Take 5 μL of each sample and transfer it to a new sample tube. Be careful not to have any bubbles in the sample tube.

[0087] (4) After removing particles from each sample, transfer 3 μL to the same centrifuge tube and mix thoroughly. Pipet 15 μL into the sample tube (be careful to avoid bubbles) for DDA library construction. Three DDA injections are performed, each requiring 5 μL.

[0088] (5) The sample tube is placed in a mass spectrometer, and the whole proteome is analyzed by mass spectrometry using the DIA model.

[0089] 2.8 Searching the Database

[0090] Spectronaut software was used to quantitatively analyze DIA data. After comparison with the mouse protein database, the full proteome map data comparison results were exported.

[0091] 2.9 Bioinformatics Statistical Analysis Methods

[0092] 2.9.1 Analysis software

[0093] This example mainly uses R software (4.2.0) for bioinformatics analysis. This example believes that the most reliable method for handling missing values ​​in the data is to define protein quantitative values ​​with excessive deviation (i.e., less than 500) as missing values ​​and perform mean imputation on missing values ​​of protein quantitative values.

[0094] 2.9.2 Sample repeatability evaluation

[0095] In this example, biological replicates were used during the protein expression extraction process. Therefore, principal component analysis (PCA) and box plot visualization were used to evaluate the protein reproducibility between samples to verify whether the experimental results of biological replicates were statistically consistent, which helps to improve the accuracy and credibility of the experiment.

[0096] 2.9.3 Whole-Proteome Difference Analysis

[0097] Based on the quality control of whole-proteome data, previous studies have shown that the power-law global analysis (PLGEW) model is beneficial for statistical analysis of proteomic data and is also one of the proteomics analysis models commonly used by our team. In this example, the PLGEM model was used to fit the quantitative results of the whole proteome and evaluate the data quality. The PLGEM model was used for differential analysis, and the differential proteins were visualized using volcano plots and heat maps based on the R language ggplot2 package.

[0098] 2.9.4 Protein functional enrichment analysis

[0099] In this example, the differentially expressed proteins were functionally annotated, and the significance P value of the enrichment test was set to be less than 0.05.

[0100] GO (Gene Ontology) enrichment analysis classifies and annotates genes to reveal the biological functions and relationships of different genes and gene sets. It mainly analyzes the functions of proteins or genes at three levels: molecular function, cellular component, and biological process.

[0101] The Kyoto Encyclopedia of Genes and Senomes (KEGG) is a systems biology database that integrates information on genomes, biochemical reactions, metabolic pathways, and more. Enrichment analysis of differentially expressed proteins can help researchers understand the functions and interactions of genes and proteins in metabolic pathways, providing important insights for studying biological processes, discovering new biomarkers, and developing drugs.

[0102] 2.10 Western blot

[0103] (1) Preparation of each sample: Use the BCA kit to detect the protein sample concentration. Take an equal amount of protein from each sample (about 30-100 μg according to the experimental needs) into a new EP tube, and make up the volume with cell lysis buffer. Add 5× Loading buffer, heat in a 95℃ water bath for 10 min, and then cool on ice.

[0104] (2) Prepare electrophoresis gel: Wash 1.5 μm thin glass plates and thick glass plates with clean water, let them dry at room temperature, and then secure them with a rack. Use pure water to check the tightness of the gel rack. First, add 10% separation gel, add anhydrous ethanol to remove bubbles, flatten the separation gel, and wait for 20-30 minutes for the separation gel to solidify. Then, add 5% stacking gel, insert the comb teeth horizontally, and wait for 20-30 minutes for the stacking gel to solidify.

[0105] (3) Loading: Clamp the glass plate with an electrophoresis tank, fill it with the prepared electrophoresis solution, remove the comb teeth horizontally to avoid the loading hole from being skewed, and add the sample to the loading hole with a pipette.

[0106] (4) Electrophoresis: Plug in the power supply and adjust the parameters: constant voltage 80V. After the sample runs through the gel, adjust the voltage to 120V. Stop the electrophoresis according to the molecular weight of the target protein and the molecular weight of the internal reference protein to prevent the sample from running off the gel.

[0107] (5) Transfer: Place 1L of pre-cooled transfer solution in the operating tray, then place a "sandwich clip" in the transfer solution with the black side down, and place the sponge and filter paper on top. Use scissors to cut a small piece of 8×5cm PVDF membrane, cutting the corner at the upper right corner to avoid the direction being reversed after transfer. After the electrophoresis is completed, peel off the thin glass plate, cut off the concentrated gel, retain the separation gel, gently separate the separation gel, and gently place the separation gel on the filter paper. Then soak the prepared PVDF membrane in methanol for 1 minute to achieve the purpose of activation. Then place the PVDF membrane on the separation gel and adjust the position so that the PVDF membrane is completely covered on the separation gel. At the same time, avoid bubbles under the PVDF membrane that affect the transfer. The whole process should be gentle to avoid breaking the separation gel. Place the "sandwich clip" into the electrotransfer tank, then pour the transfer solution from the operating tray into the electrotransfer tank to ensure that the PVDF membrane is completely immersed in the transfer solution. Place an ice box in the remaining empty space to ensure the safety of electrotransfer. Adjust the parameters: constant voltage 100V, current 235mA, and start electrotransfer after 120 minutes.

[0108] (6) Blocking: Prepare 5% blocking solution, place the PVDF membrane after transfer into a small box containing TBST (containing Tween) and wash it twice. Remove TBST (containing Tween), add the prepared 5% blocking solution until the blocking solution submerges the PVDF membrane, and shake it slowly on a shaker for 1 hour.

[0109] (7) Incubation of primary antibody: After blocking, remove the blocking solution, add TBST (containing Tween) to wash the PVDF membrane and shake it on a shaker for 5 minutes, remove the TBST (containing Tween), and repeat this washing step 3 times to remove all the blocking solution. Soak the PVDF membrane in the prepared primary antibody solution and then shake it slowly on a shaker at 4°C overnight.

[0110] (8) Incubation with secondary antibody: Recover the primary antibody solution, add TBST (containing Tween) to wash the PVDF membrane, shake it on a shaker for 5 minutes, remove the TBST (containing Tween), repeat this washing step 3 times, add the prepared secondary antibody, soak the PVDF membrane in the secondary antibody solution, and shake it slowly on a shaker for 1 hour.

[0111] (9) Development: Remove the secondary antibody solution, add TBST (containing Tween) to wash the PVDF membrane, shake it on a shaker for 5 minutes, remove the TBST (containing Tween), repeat this washing step 3 times, bring the prepared luminescent solution, tweezers, paper towel and PVDF membrane to the front of the biomolecular imaging instrument, gently pick up the PVDF membrane with tweezers, place it face up on the paper, absorb as much residual TBST (containing Tween) as possible, and then place it on the biomolecular imaging instrument, drop the luminescent solution on the target protein band to develop and save the results.

[0112] 3. Experimental Results and Statistical Analysis

[0113] P < 0.05 and Foldchange > 1.5 were considered statistically significant. All data analyses were performed in R 4.2.0 software.

[0114] 4. Serum NGP in patients with hepatectomy predicts postoperative complications

[0115] Serum samples were obtained from patients undergoing liver resection. Postoperative complications were recorded and classified according to the Clavien-Dindo classification, with grade III-V severe complications considered positive.

[0116] Testing serum samples

[0117] b1) Quantitative chip drying

[0118] Take the quantitative chip out of the box, place it at room temperature for 20-30 minutes, open the packaging bag, remove the sealing strip, and then place the quantitative chip in a desiccator or at room temperature to dry for 1-2 hours.

[0119] b2) Preparation of standard products

[0120] b21) Add 500 μL of sample diluent to the standard mixture vial to reconstitute the standard mixture. Before opening the vial, quickly centrifuge and gently pipette up and down to dissolve the powder. Label this vial Std 1.

[0121] b22) Label 6 clean centrifuge tubes as std2, std3, std4, std5, Std6, and Std7, and add 200 μl of sample diluent to each tube.

[0122] b23) Draw 100 μL of the sample diluent from the centrifuge tube labeled Std 1 and add it to the centrifuge tube labeled Std 2 and mix gently. Then, draw 100 μL of the sample diluent from the centrifuge tube labeled Std 2 and add it to the centrifuge tube labeled Std 3. And so on. Continue with this gradient dilution until the tube labeled Std 7 is reached to obtain the standard solution.

[0123] b24) Transfer 100 μL of the standard solution from the centrifuge tube labeled Std7 to another new centrifuge tube labeled CVTRL as a negative control.

[0124] b3) Operational procedures of quantitative chip

[0125] b31) Add 100 μL of sample diluent to each well of the quantitative chip, incubate on a shaker at room temperature for 1 hour, and seal the quantitative chip.

[0126] b32) Remove the sample diluent from each well, take 80 μL of the standard solution from the centrifuge tube labeled CNTRL and add it to the well together with the serum sample, and incubate at 4°C overnight.

[0127] b33) Clean the quantitative chip. First, wash with 1× Wash Buffer I (250 μL per well) for 10 washes, vortexing for 10 seconds each time (high intensity). Dilute 20× Wash Buffer I with deionized water. Then, clean the channel with 1× Wash Buffer II (250 μL per well) for 6 washes, vortexing for 10 seconds each time (high intensity). Dilute 20× Wash Buffer II with deionized water.

[0128] b34) Incubation of IgG antibody against NGP protein: Centrifuge the tube containing IgG antibody against NGP protein, then add 1.4 mL of sample diluent, mix well and centrifuge again quickly, then add 80 μL of IgG antibody against NGP protein to each well and incubate on a shaker at RT for 2 hours.

[0129] b35) Cleaning, same as step b33).

[0130] b36) Cy3-streptavidin incubation: Centrifuge the tube containing Cy3-streptavidin, then add 1.4 mL of sample diluent, mix thoroughly, and quickly centrifuge again. Add 804 mL of Cy3-streptavidin to each well. Wrap the quantitative chip with aluminum foil and incubate in the dark. Incubate on a shaker at RT for 1 hour.

[0131] b37) Cleaning, same as step b33).

[0132] b38) Fluorescence detection: use a laser scanner to scan the signal, using Cy3 or green channel.

[0133] b39) QAHI-CUST data analysis software was used to analyze the data and obtain the NGP protein content in each serum sample.

[0134] The term "ROC curve" or "ROC diagram" used in the present invention refers to a graphical curve showing the performance of a binary classifier system as a function of its discrimination threshold. This curve is created by plotting the true positive rate versus the false positive rate under various threshold settings. The true positive rate is also referred to as sensitivity. The false positive rate is calculated as 1-specificity. Therefore, the ROC curve is a graphical display of the true positive rate versus the false positive rate (sensitivity vs (1-specificity)) within a range of cutoff values ​​and a method for selecting the best cutoff value for clinical use. Accuracy is expressed as the area under the ROC curve (AUC), which provides a useful parameter for comparing test performance. An AUC close to 1 indicates that the test is highly sensitive and highly specific, while an AUC close to 0.5 indicates that the test is neither sensitive nor specific.

[0135] 5.1 Quality Control of Whole Proteome Mass Spectrometry Data

[0136] Since the quality of proteomic data affects subsequent analysis and verification, it is necessary to perform quality control on the proteomic data. In this example, the quality of proteomic data is evaluated by data distribution comparison and PCA analysis. Figure 1 As shown, the horizontal axis represents the sample name, and the vertical axis represents the Log10-transformed intensity value. The box colors represent different groupings. Horizontal comparison provides a rough indication of the degree of dispersion of data distribution within and between groups. Sample means are on the same horizontal line, and protein expression distribution trends are similar, indicating good sample quality. Principal component analysis was performed based on the relative quantitative values ​​of all samples, and a visual PCA plot was drawn. The horizontal and vertical axes show the explanatory power of PC1 and PC2, with larger values ​​indicating higher explanatory power. Figure 2 Shows that replicates within each group tend to cluster together.

[0137] In this example, proteins with PLGEM model differential analysis P < 0.05 and Foldchange > 1.5 were defined as differential proteins (liver injury group vs. control group; Different Proteins, DEPs). In the whole proteome data, there were 142 differential proteins in the T1 group compared with the control group, including 81 up-regulated differential proteins and 61 down-regulated differential proteins; there were 298 differential proteins in the T2 group compared with the control group, including 141 up-regulated differential proteins and 157 down-regulated differential proteins. Figure 3 Volcano map and Figure 4 The heat map fully demonstrated that the screened DEPs can clearly distinguish the control group from the liver injury group.

[0138] 5.2 Proteome-wide differential protein enrichment analysis

[0139] In order to further explore the biological processes and cell functions affected by liver damage, GO enrichment analysis was performed on the differential proteins, such as Figure 5 As shown in the results, compared with the control group, the biological processes (BP) in the T1 group were enriched in neutrophil aggregation, endothelial cell signaling pathways, positive regulation of macrophage activation, negative regulation of epidermal cell apoptosis, and regulation of keratinocyte proliferation. Compared with the control group, the biological processes in the T2 group were enriched in induction of bacterial coagulation, endothelial cell differentiation, regulation of TH1 immune response, fibrinolysis, and acute phase response.

[0140] In addition, the KEGG enrichment analysis results of differential proteins suggested that the differential proteins in the T1 / C group were significantly clustered in tumor transcriptional dysregulation, acute myeloid leukemia, hematopoietic cell line, tuberculosis and IL-17 signaling pathways, while the differential proteins in the T2 / C group were significantly clustered in intestinal IgA synthesis immune network, sphingomyelin biosynthesis, complement and coagulation cascade, Staphylococcus aureus infection and mineral element absorption signaling pathways, such as Figure 6 shown.

[0141] In summary, the results of differential protein enrichment analysis showed that the pathophysiological mechanism of liver injury is particularly complex, which affects the treatment and prognosis of liver injury.

[0142] 5.4 Western Blot Verification of Mass Spectrometry Data Accuracy

[0143] Western Blot was used to further verify the expression of NGP in liver tissue. The expression of NGP in liver tissue at the whole protein level showed that NGP was upregulated after liver ischemia-reperfusion. Figure 7 shown.

[0144] In summary, the present invention uses mass spectrometry to obtain liver injury-related proteomic data and identifies biological signaling pathways associated with liver injury. Furthermore, it identifies a close correlation between NGP and liver injury. Western blot analysis confirms that NGP expression is upregulated in the liver injury group, consistent with the results of mass spectrometry analysis. This invention pioneers the application of these biomarkers in the prognosis of liver injury, providing a new target for the study of drugs to treat liver injury. This invention has significant clinical, scientific, and drug translational value.

[0145] The diagnostic effect of the model is as follows Figure 8 As shown in Figure 2, the AUC of serum NGP level was 0.787 one day after surgery. Figure 8 As shown in the results, the sensitivity and specificity of serum NGP level at a specific cutoff of 1 day after surgery were 64.4% and 72.9%, respectively. This indicates that serum NGP level at 1 day after surgery has a good predictive effect on postoperative complications after liver resection.

[0146] The preferred specific implementation modes and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation modes and embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the concept of the present invention.

Claims

1. A proteomics-based biomarker for liver ischemia-reperfusion injury, characterized in that: The biomarker is neutrophil granule protein (NGP).

2. The use according to claim 1, characterized in that The biomarkers are detected by proteomics analysis on samples, and the samples are from mouse liver tissue.

3. Application of neutrophil granule protein (NGP) in the preparation of reagents, kits, microarrays or biochips for predicting postoperative complications in clinical liver resection patients.

4. The use according to claim 3, characterized in that: The neutrophil granule protein is human serum neutrophil granule protein.

5. The use according to claim 3, characterized in that The clinical postoperative complications of liver resection patients are severe complications of grade III-V according to the Clavien-Dindo classification.

6. The use according to claim 3, characterized in that The clinical postoperative complications of liver resection patients include bleeding, liver failure, abdominal infection, bile leakage, pleural effusion and pulmonary complications, and ascites.

7. The use according to claim 3, characterized in that The reagent or kit includes immunoglobulin G antibody against neutrophil granule protein.

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