Detection of Salivary Protein Biomarkers in a Primary Sjögren Syndrome Murine Model
Salivary biomarkers related to saliva secretion disorder were identified in a primary Sjögren syndrome (pSS)pSS murine model, NOD/ShiLtJ mouse, using differential proteomic analysis. We used 14-week-old female NOD/ShiLtJ mice and age- and sex-matched BALB/c mice as controls. 30 stimulated saliva samples and mixed them into six protein pools (three pSS pools and three control pools) for differential proteomic analysis.After Nano-high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) separation, 98.14% (69,276/69,276) spectra were used for protein identification. We identified 1,101 proteins from saliva; 68.12% (750/1,101) were detected both in NOD/ShiLtJ and BALB/c mice .Two mice strains showed a similar salivary protein spectrum. Differentially expressed proteins were determined by applying Mann-Whitney test, corrected by Benjamini-Hochberg method at a significance level of p<0.03173 and fold change of >1.5.
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Sample preparation and Tandem Mass Tag (TMT) labeling Every five samples comprising 100 μg protein each were mixed and pooled. Three pSS and three control pools were prepared and diluted to 100 μL with 8 M urea in 0.1 M Tris-HCl (pH 8.0).Proteins were subjected to tryptic digestion with trypsin at 1:50 (m/m) concentration and the resultant peptide mixture was labeled using chemicals from TMT 6plex reagent kit, according to the manufacturer’s protocol. In pSS group, three NOD/ShiLtJ samples were labeled by TMT with reporters at m/z of 126.1, 127.1, and 128.1. In the healthy control group, three BALB/c samples were labeled by another set of TMT with reporters at m/z of 129.1, 130.1, and 131.1. Another technical replicate was obtained for repeating measurements on the same saliva sample. The samples were combined and dried in vacuum. The peptide mixture was redissolved in buffer A (20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide) and fractionated with high pH separation using Ultimate 3000 system (ThermoFisher scientific, MA, USA) connected to a reverse-phase column . High pH separation was performed using a linear gradient from 5% to 45% B in 40 min (B: 20 mM ammonium formate in 80% acetonitrile, pH 10.0 adjusted with ammonium hydroxide). The column was re-equilibrated at initial conditions for 15 min. The column flow rate and temperature were maintained at 1 mL/min and 30°C, respectively. Fifteen fractions were collected and each fraction was dried in a vacuum concentrator. The fractions were resuspended in 30 μL solvent C (water with 0.1% formic acid), separated by nanoLC, and analyzed by on-line electrospray tandem mass spectrometry. The experiment was performed on a Nano Aquity UPLC system connected to a quadrupole Orbitrap mass spectrometer (Q-Exactive)equipped with an on-line nano-electrospray ion source. A total of 5 μL peptide sample was loaded onto the trap columnat a flow rate of 10 μL/min for 3 min and subsequently separated on the analytical column with a linear gradient from 2% to 40% D (ACN with 0.1% formic acid) in 100 min. The column was re-equilibrated at initial conditions for 15 min. The column flow rate and temperature were maintained at 300 nL/min and 40°C. The electrospray voltage of 1.9 kV versus the inlet of the mass spectrometer was used.The fusion mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350-1550) were acquired with a mass resolution of 120 K, followed by sequential high energy collisional dissociation (HCD) MS/MS scans with a resolution of 30 K. The isolation window was set as 1.6 Da. AGC target was set as 400,000. MS/MS fixed first mass was set at 110. In all cases, one microscan was recorded using dynamic exclusion of 45 s.