3.1 Patient and sample collection
Sural nerve samples for the experimental group were obtained from patients who met the criteria for pSS peripheral neuropathy, while those for the control group were chronic inflammatory demyelinating polyneuropathy. Patients with diabetes mellitus, systemic autoimmune diseases, malignant tumors, severe hepatic or renal insufficiency were excluded. All nerve samples were collected from remaining clinical nerve samples within three years. Sural nerve samples were collected from three cases in each group and numbered accordingly. The experimental group was labeled as VN-1 to VN-3, and the control group was labeled as CN-1 to CN-3.
3.2 Compliance with Ethical Standards
Study protocols were reviewed and approved by the Medical Ethics of committee of Nanfang Hospital of Southern Medical University(No.NFEC-2023-170). Participants provided and approved written informed consent.
3.3 Sample preparation and PCT-assisted analysis and enzymatic hydrolysis
The frozen samples were removed and placed in a 1.5 ml centrifuge tube. Add 1ml of 70% ethanol to each tube, clean the tissue for 30s at 800rpm and discard the supernatant; add 1ml of ultra-pure water to each tube and repeat the above; add 1ml of 70% ethanol to each tube, clean the tissue at 800rpm for 5min, discard the supernatant and repeat. Then add 1ml of 85% ethanol and repeat the above procedure; add another 1ml of 100% ethanol and repeat.
Each sample was mixed with 30 μL of pyrolysis liquid containing 6M urea, 2M thiourea, and 100mM ammonium bicarbonate (ABB) at a pH of around 8. To initiate the reaction, 5μL of DNase was added, and the samples were subjected to high-pressure reactions in a PCT instrument. The reaction system involved 60 cycles of alternating 25s 45000psi high pressure and 10s normal pressure with a reaction temperature of 30℃. After completion of the reaction, TCEP and IAA were added to each tube at final concentrations of 10mM and 40mM, respectively. The reaction was carried out for 30min under light-avoiding conditions at 25℃ and 800rpm. To achieve a final urea concentration of 1.6M, 100 μL of 0.1M ammonium bicarbonate solution was added followed by the addition of Lys-C at a ratio of 1:40 (enzyme: protein, w/w). The reaction was carried out using the PCT instrument for 45 cycles, involving 50s 20000psi and 10s atmospheric pressure alternating cycles, with a temperature of 30℃. To maintain a urea concentration of 1M, 0.1M ammonium bicarbonate solution was added before the addition of a corresponding amount of pancreatic enzyme at a Trypsin: protein mass ratio of 1:50. The sample was subjected to a second series of reactions in the PCT instrument programmed with 90 cycles, involving 50s 20000psi and 10s atmospheric pressure alternating cycles, and a temperature of 30℃. After completion of the reaction, 10% TFA solution was added to each tube to terminate pancreatic enzyme digestion, with a pH of 2-3.
3.4 Peptide purification and quantitative
The samples were initially treated with 200 μL methanol and centrifuged at 1200 rpm for 30 s, repeated three times. Next, an activated C18 microcolumn was used, and 200 μL 80% ACN/0.1% TFA was added before being centrifuged at 1400 rpm for 1 min, repeated three times. After that, 200 μL 2% ACN was added, and the mixture was centrifuged at 1400 rpm for 2 min, repeated three times. The resulting peptide solution (approx. 160 μL) was added to the C18 column, centrifuged at 2000 rpm for 3 min, and repeated twice. The peptides adsorbed on the column were then cleaned using 200 μL wash buffer (2% ACN), centrifuged at 1600 rpm for 2 min, and repeated six times. Finally, the peptide was eluted using 150 μL 80% ACN/0.1% TFA, centrifuged at 1600 rpm for 3 min, repeated once, and the two fractions were combined and vacuum-dried for future use. The peptide was subsequently redissolved in a 0.1% formic acid/2% acetonitrile solution, sonicated on ice for 5 min, and quantified using NanoDrop 1000. The quantitative results can be found in Appendix 1.
3.5 DDA library construction
All samples underwent enzymatic digestion and were subsequently combined with an equal amount of peptide segments. The mixture was then subjected to separation by a C18 column (Agilent Zorbax Extend-C18 narrow diameter column, 2.1×150mm, 5μm) at a rate of 300μL/min using an Agilent 1100 HPLC system. Gradient elution was achieved using mobile phases A (2% ACN, pH adjusted to 10.0 with ammonia) and B (90% ACN, pH adjusted to 10.0 with ammonia). The solvent gradient was set as follows: 0-10 min, 2% B; 10-10.01 min, 2-5% B; 10.01-37 min, 5-20% B; 37-48 min, 20-40% B; 48-48.01 min, 40-90% B; 48.01-58 min, 90% B; 58-58.01 min, 90-2% B; 58.01-63 min, 2% B. The eluent was detected at UV210nm and 280nm. Starting from 10min, one tube was collected every 1min, and a total of 10 components were collected and drained by vacuum freeze-drying. For subsequent analysis, the iRT standard (Biognosys, ThermoFisher) was mixed with each sample in a volume ratio of 1:10 and used as an internal standard. The iRT standard was prepared by dissolving it in a solution with a concentration of 10× and stored at 2-8℃.
For construction of the transition library, proteomic analysis was carried out using the EASY-nLC 1200 liquid chromatography system coupled to a Q Exactive HF mass spectrometer (ThermoFisher) operating in data-dependent acquisition (DDA) mode. The peptides were separated on PolySULPHOETHYL A liquid chromatography column (2cm × 9.4mm, 5μm) with a linear gradient of 90 minutes. Mobile phase A was a 0.1% FA aqueous solution, while mobile phase B was a mixture of 0.1% FA, 80% ACN, and 20% water. The detailed solvent gradient was as follows: 8%-25% B in 0-60 minutes, 25%-45% B in 60-79 minutes, 45%-100% B in 79-80 minutes, and 100% B in 80-90 minutes.
The Q Exactive HF mass spectrometer operated in positive ion mode and performed a full MS scan from 350-1650 m/z at a resolution of 120,000 with an automatic gain control (AGC) set to 3e6 and a maximum injection time of 100 ms. An MS2 scan of 200-2000 m/z was performed at a resolution of 30,000 with an AGC target of 2e5, a maximum injection time of 80 ms, an isolation window of 1.4 m/z, and a normalized collision energy (NCE) of 27%.
3.6 MS analysis of DIA mode for each sample
The enzymatic hydrolysis peptides of each sample were mixed in a 1:10 volume ratio with iRT and injected into the EASY-nLC1200 liquid chromatography system for use with the Q Exactive HF mass spectrometer operating in DIA mode. The liquid conditions were consistent with the library construction of the DDA model. For DIA acquisition, the Full MS resolution was set at 120,000 and the MS2 resolution at 30,000. The m/z range was 350-1250, and the isolation window was 26 m/z. The full-scan AGC target was set at 3e6, and the injection time was 100 ms. DIA settings included an NCE of 28%, a target value of 1e6, and a maximum injection time set to automatic.
3.7 Validation study by PRM
The protein in the sample was isolated and the protein concentration was determined using bicinchoninic acid (BCA). Subsequently, all proteins were enzymatically digested and the resulting peptides were desalted and are now prepared for analysis using the machine.
The libraries were constructed using an equal combination of all samples, and mass spectrometry was employed for MS analysis. The iRT standard peptide solution was mixed with the tested sample at a volume ratio of 1:10 to prepare for LC-MS/MS analysis. To minimize the retention time bias of the fluid mass system, prescan correction was performed initially.
The chromatographic separation and full scan conditions remained the same as mentioned above. For PRM, a resolution of 30,000 (at an isolation window of 1.2 m/z) was set, along with an AGC target value of 2e5, a maximum accumulation time of 100 ms, and an HCD energy of 28. Following offline analysis utilizing SpectroDive software, each sample's identification was manually verified. Biased peptide fragments were adjusted, and the target peptide fragment's quantitative outcomes were obtained at all times. Software built-in mean peptide quantity was used to calculate quantitative values of simultaneous proteins for statistical analysis between groups.