Patient recruitment and follow-up
All participants were recruited at Capital Medical University Xuanwu Hospital from 2019 to 2020. The patient cohort was a part of the COPSDAVF study (ClinicalTrials.gov, NCT03192800), which is described in detail elsewhere . In brief, patients who were diagnosed by DSA and received lumbar puncture before surgery were included in this study. The exclusion criteria were as follows: (1) ≤ 18 years of age; (2) history of lumbar surgery, stroke, demyelinating, and other neurological diseases; (3) upper cervical spinal cord or medulla oblongata oedema, incurring the risk of cerebral herniation; and (4) MRI or DSA revealed that the fistula drained into the horsetail, especially below the L2 vertebral level. Two researchers (Y. M. and C. Y.) independently screened clinical information to complete patient recruitment. We used modified Aminoff and Logue's Scale (mALS)  and modified Denis Pain and Numbness Scale (mDS)  to evaluate the patient's neurological function, and the combined scores (CS) were equal to mALS scores plus mDS scores (for the detailed items, see Additional file 2: Table S1). Scheduled follow-up was 3 months, 6 months, and 12 months. Patients were followed via outpatient visits or telephone interviews, and those in the validation cohort had at least 6 months of follow-up. The improvement of CS was calculated as follows: 6-month follow-up CS minus presurgical CS. One patient in the discovery cohort was lost to follow-up due to incorrect contact information. The control group included participants who underwent artificial joint replacement surgery by spinal anaesthesia with no history of neurological diseases. The sample size of the validation cohort was calculated as 10 in the patient group and 6 in the control group, with the hypothesis of AUC = 0.9. This study was approved by the Ethics Committee of Xuanwu Hospital and all subjects signed informed consent forms. The overall workflow is shown in Fig. 1.
Five to ten millilitres of cerebrospinal fluid (CSF)was collected by lumbar puncture. Two millilitres of CSF was sent to the hospital laboratory for routine examination including cell count and differential (XN-2000-Haematology-Analyser-Sysmex), total protein, and glucose (7600 Automatic Biochemical Analyser Hitachi). Meanwhile, paired 4 ml blood was collected using Vacutainer CPT tubes (BD Biosciences) containing EDTA. Samples were transferred to the biobank of our hospital at 4 ℃ within 2 h. Then, CSF and whole blood samples were centrifuged at 1500 r/min and 3000 r/min, respectively, for 10 min. The supernatant was collected in 0.5 ml aliquots in polypropylene tubes (Corning, 430,659) and stored at –80 °C.
In the discovery cohort, the 8 samples of each group were mixed and aliquoted into 4 centrifuge tubes (see Fig. 1). The mixed samples were lysed in buffer containing 4% sodium dodecyl sulfate and 0.1 M Tris–HCl (pH 7.6). The protein concentration was determined by the BCA protein assay kit (Thermo Scientific, Rockford, IL). The proteins (100 μg) were incubated with 100 mM DTT at 37 ℃ for 1 h. After incubation, the lysates were transferred to a centrifugal filter (Microcon YM-30, EMD Millipore Corporation, Billerica, MA), and replaced with 200 μl UA (8 M urea, 100 mM Tris. Cl pH 8.5) twice. After the buffer was replaced, proteins within UA were alkylated with 55 mM iodoacetamide (IAA, Sigma–Aldrich, Saint Louis, MO) in a dark at room temperature for 15 min. The UA buffer was subsequently replaced with 0.1 M triethylammonium bicarbonate (TEAB, Sigma–Aldrich, Saint Louis, MO) and digested with trypsin (Promega, Madison, WI) (1:50 (w: w)) at 37 ℃ overnight. The desalting of resultant tryptic peptides was conducted by StageTips and stored at − 20 °C.
LC–MS/MS and data analysis
The peptides (2 μg) were resolubilized in 0.1% formic acid (FA) and analysed by a mass spectrometer (Orbitrap Fusion™ Lumos™ Tribrid™, Thermo Scientific, Rockford, IL Waltham, MA) coupled to an Easy-nLC 1200. The samples were run with mobile phases containing buffer A (0.1% FA) and buffer B (80% ACN, 0.1% FA). The peptides were separated by a capillary analytic C18 column (length: 25 cm, inner diameter: 150 μm, particle diameter: 1.9 μm) in a 180-min nonlinear gradient at a flow rate of 600 nl/min. For each three-second cycle, the full MS scan was acquired in the Orbitrap at a resolution of 120,000 with an automatic gain control (AGC) target of 5 × 105, and higher-energy collisional dissociation (HCD, collision energy: 30%) was used to fragment these precursors. An MS/MS scan was performed in the IonTrap (AGC = 3 × 104). MaxQuant (version 1.6) software was used for database search and label-free quantitative analysis. The Homo sapiens proteome sequence database was downloaded from the Uniprot website . The parameters of the database search were set as follows: type: standard; multiplicity: 1; the protease used for protein digestion: trypsin; label-free quantification: LFQ; the minimum score for unmodified peptides: 15. All other parameters were default values. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner repository with the dataset identifier PXD030479.
The data analysis of this data was mainly run in R (version 3.6.3). We defined significantly differentially expressed proteins (DEPs) as those with a fold change > 1.5 (up- and down-regulated) and P < 0.05. The DEPs were visualized by volcano plot, cluster heatmap, and Venn plot via the ggplot2 package. The annotation and enrichment of GO terms and KEGG pathways was achieved through the clusterProfiler package and org.Hs.e.g.db package . The protein–protein interaction (PPI) annotation of DEPs was obtained from the STRING database (Version 11.5, string-db.org). The PPI subcluster and top10 hub genes were extracted via MCODE and the CytoHubba plugin of Cytoscape software (version 3.8.2).
Seventeen cases and nine controls were included in the validation cohort. The experimental performers (Z.S. and A.T.) were blinded to group information. Sources of the ELISA kit and dilution fold of CSF samples were listed as follows: LBP (2× dilution; Human, E-EL-H6108, elabscience, China), APP (2× dilution; Human, E-EL-H1216c, elabscience, China), C4BPA (2× dilution; Human, CSB-E11170h, cusabio, China), APOB (200× dilution; Human, E-EL-H0464c, elabscience, China), C1QA (10× dilution; Human, CSB-EL003637HU, cusabio, China), C4 (50× dilution; Human, CSB-E08705h, cusabio, China), and MASP2 (Neat; Human, CSB-E17966h, cusabio, China). All samples were set in triplicate wells. According to the manufacturer’s manual, standards or samples (100 µl each) were added to 96-well plates and incubated for 90 min at 37 °C. After discarding the liquid from each well, a biotinylated antibody working solution (100 µl) was added to each well and incubated at 37 °C for 1 h. The plates were washed 3 times. Subsequently, 100 μl of the horseradish peroxidase (HRP)-streptavidin conjugate was added to each well and incubated for 30 min at 37 °C. The plates were then washed 5 times, and 90 μl of substrate solution was added to each well and incubated at 37 °C in the dark for 15 min. The reaction was stopped by adding 50 µl of stop solution and the absorbance value was immediately measured at 450 nm (Multiskan FC, Thermo Scientific).
All statistical analyses and visualization were performed in R (version 3.6.3) or GraphPad Prism (version 8.0.1). The sample size was estimated by PASS (version 15.0.5). The quantitative results of ELISA were normalized to the total protein in each patient. An independent samples t test was used to compare the means of continuous normally distributed data. The Chi-squared test was adopted for the comparison of the categorical variables, such as sex. When the data showed a non-normal distribution, Wilcoxon rank-sum tests were applied. The association of target proteins with the clinical factors was visualized by scatter plot and fitted by simple linear regression. Receiver operating characteristic (ROC) curves were generated to investigate the diagnostic performance of biomarkers. All statistical tests were two-tailed. p or adj. p < 0.05 indicated statistical significance.