PRIDE Assigned Tags:Biomedical Dataset
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Salivary Protein Biomarker Detection of Saliva Secretion Disorder in a Primary Sjögren Syndrome Murine Model
Decreased salivary flow is one of the most common symptoms in primary Sjögren syndrome (pSS). The present study was designed to identify a salivary protein biomarker related to saliva secretion disorder in NOD/ShiLtJ mice, a pSS primary murine model. The stimulated saliva was collected from NOD/ShiLtJ and BALB/c mice as controls for differential proteomic analysis.
Sample Processing Protocol
Fifteen 14-week-old female NOD/ShiLtJ mice and 15 age- and sex-matched BALB/c mice were fasted for a minimum of 12 h with water ad libitum. Under 60 mg/kg pentobarbital sodium intraperitoneal anesthesia, stimulated saliva was collected and weighed from the oral cavity for 15 min, starting at 5 min after 0.05-mg/kg pilocarpine and 0.1-mg/kg isoproterenol subcutaneous injection. The protein concentrations in 30 stimulated saliva samples collected from the NOD/ShiLtJ and BALB/c mice were determined with the BCA Protein Assay Kit (CWBIO, Beijing, China). Every five samples, which contained 100-μg protein each, were mixed into one pool. Finally, 3 pSS and 3 control pools were prepared and diluted to 100 μl by 8-M urea in 0.1-M Tris-HCl (pH 8.0). Subsequently, 11-μl 1-M DTT was added and incubated at 37°C for 1 h. The samples were applied to the 10-kDa ultrafiltration tube (Millipore, MA, USA) and centrifuged at 14000g for 15 min. Subsequently, 100-μl 55-mM iodoacetamide was added and incubated for 20 min at room temperature. Thereafter, 50-mM triethylammonium bicarbonate (TEAB) was used as exchange buffer. Proteins were the tryptic digested with trypsin (Promega, WI, USA), and the resultant peptide mixture was labeled using chemicals from the TMT 6plex reagent kit (Pierce Biotechnology, IL, USA). The samples were combined and dried in vacuum. The peptide mixture was redissolved and fractionated by high-pH separation using the Ultimate 3000 system (ThermoFisher Scientific, MA, USA) connected to a reverse-phase column (Waters Corporation, MA, USA). Fifteen fractions were collected. The fractions were resuspended with 2% (v/v) acetonitrile/0.1% (v/v) formic acid in water and separated using the Aquity UPLC system (Waters Corporation, Milford, MA) connected to a Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A 5-μl peptide sample was loaded onto the trap column with a flow of 10 μl/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm) with a linear gradient, from 2% D to 40% D in 100 min. The 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. The AGC target was set as 400,000.
Data Processing Protocol
All MS/MS samples were analyzed using Mascot v2.5.1 (Matrix Science, London, UK), which was searched with a fragment and parent ion mass tolerance of 0.050 Da and 7.0 ppm, respectively. Scaffold v4.7.2 (Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could achieve an FDR of <1.0% by the scaffold local FDR algorithm. Protein identifications were accepted if they contained at least two identified peptides. Scaffold Q+ v4.7.3 (Proteome Software Inc., Portland, OR, USA) was used to quantitate peptide and protein identifications. Normalization was performed iteratively on intensities, as described by Oberg et al.
Qian Tao, 1 Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University
Qian Tao, Guanghua school of stomatolgy, Hospital of stomatology, Sun Yat-sen university ( lab head )
Liang P, Zhu W, Lan T, Tao Q. Detection of salivary protein biomarkers of saliva secretion disorder in a primary Sjögren syndrome murine model. J Pharm Biomed Anal. 2018 154:252-262 PubMed: 29558726