DOI: 10.25345/c5gq6rd1b
title: MassIVE MSV000094456 - Proteomic analysis of calcific aortic valve stenosis in the rabbit high fat vitamin D diet model
title: Proteomic analysis of aortic valve tissue by LC-MS/MS. A total of N=11 samples were analyzed: N=5 cases (E28, E30, E31, E32, E33) and N=6 controls (C38, C39, C40, C41, C42, C43). Sample preparation for the proteomics analysis was performed with the GeLC-MS method. Briefly, 10 microgram of each sample was analyzed in SDS-PAGE (5% stacking, 12% separating). Electrophoresis was stopped when samples just entered the separating gel. Gels were fixed with 30% methanol, 10% acetic acid for 30 min followed by 3 washes with water (3x10min) and stained with coomassie colloidal blue overnight. Excess of stain was washed with water (3x10min washes). Each band was excised from the gel and further sliced to small pieces (1-2mm). Gel pieces were destained with 40% Acetonitrile, 50mM NH4HCO3 and then reduced with 10mM DTE in 100mM NH4HCO3 for 20min RT. After reduction, samples were alkylated with 54mM Iodoacetamide in 100mM NH4HCO3 for 20min RT in the dark. Samples were then washed with 100mM NH4HCO3 for 20 min, RT, followed by another wash with 40% Acetonitrile, 50mM NH4HCO3 for 20min, RT and a final wash with ultrapure water under the same conditions was performed. Gel pieces were dried in a centrifugal vacuum concentrator (speed vac) and trypsinized overnight in the dark, RT, by adding 600ng of trypsin per sample (trypsin stock solution: 10ng/microliter in 10mM NH4HCO3, pH 8.5). Peptides were extracted after incubation with the following buffers: 50mM NH4HCO3 for 15min, RT followed by two incubations with 10% Formic Acid, Acetonitrile (1:1) for 15 min, RT. Peptides were eluted in a final volume of 0.6mL and filtered with PVDF filters (Merck Millipore) before dried in a centrifugal vacuum concentrator (speed vac). Dried peptides were reconstituted in mobile phase A buffer (0.1% formic acid, pH 3) and processed with LC-MS/MS analysis. Each sample was reconstituted in 10 microliters loading solution composed of 0.1 % v/v formic acid. A 5 microliters volume was injected and loaded for the LC-MS/MS analysis. Mobile phase (A) was composed of 0.1 % formic acid and mobile phase (B) was composed of 100% acetonitrile, 0.1% formic acid. The peptides were eluted under a 240 minute gradient from 2% (B) to 80% (B). Flow rate was 300 nL/min and column temperature was set at 35C. Gaseous phase transition of the separated peptides was achieved with positive ion electrospray ionization applying a voltage of 2.5 kV. For every MS survey scan, the top 10 most abundant multiply charged precursor ions between m/z ratio 300 and 2200 and intensity threshold 500 counts were selected with FT mass resolution of 70,000 and subjected to HCD fragmentation. Tandem mass spectra were acquired with FT resolution of 35,000. Normalized collision energy was set to 33 and already targeted precursors were dynamically excluded for further isolation and activation for 30 sec with 5 ppm mass tolerance. Raw files were analysed with Proteome Discoverer 1.4 software package (Thermo Finnigan), using the Sequest search engine and the Uniprot Oryctolagus cuniculus reviewed database, downloaded on March 19, 2021. The search was performed using carbamidomethylation of cysteine as static and oxidation of methionine as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05 Da were allowed. False discovery rate (FDR) validation was based on q value: target FDR: 0.05.Label free quantification was performed by utilizing the precursor ion area values exported from the total ion chromatogram as defined by the Proteome Discoverer 1.4 software package. Output files from Proteome Discoverer were processed with R programming language for statistical computing (version 4.0.3). Raw protein intensities for each sample were subjected to normalization according to X' = X/Sum(Xi) x 10e6. Statistical analysis was performed with the non-parametric Mann-Whitney test. Proteins with ratio &gt;1.5 (up-regulated) or &lt;0.67 (down-regulated) were considered differentially expressed.