DOI: 10.25345/c5hh6cj1t
title: MassIVE MSV000096062 - Analysis of changes in the proteome of VIC and VEC cells co-cultured using SILAC medium
title: Here we present data from a pilot experiment for co-culturing valve interstitial cells (VIC) and valve endothelial cells (VEC) in SILAC medium and analyzing changes in VIC and VEC proteome to study osteodifferentiation in the aortic valve. Protein isolation. The RIPA solution with cells was exposed to ultrasound (2 times for 20 minutes with intermediate incubation on ice for 10 minutes) for protein extraction. Then protein was precipitated by 4 volumes of iced cold acetone (LC-MS Grade; LiChrosolv) and washed with methanol (LC-MS Grade; LiChrosolv). Protein pellet was air-dried and resuspended in 8 M urea (Sigma Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate (Sigma Aldrich, St. Louis, MO, USA). Sample preparation for shotgun proteomics. Protein concentrations were measured by Qubit 2.0 fluorimeter (Thermo Fisher Sci, Waltham, MA, USA) with QuDye Protein Quantification Kit (Lumiprobe, Moscow, Russia). 15 mkg of each sample were used for further LC-MS/MS analysis. Samples were incubated for 1 h at 37C with 5 mM DTT (Sigma Aldrich, St. Louis, MO, USA) with subsequent incubation in 15 mM iodoacetamide (Sigma Aldrich, St. Louis, MO, USA) for 30 min in the dark at room temperature, followed by quenching with 5 mM DTT. The samples were diluted with seven volumes of 50 mM ammonium bicarbonate and incubated for 17 h at 37C with Trypsin Gold in ratio 1:50 (Promega, Madison, WI, USA). Tryptic peptides were desalted by solid-phase extraction using stage-tips (Rappsilber et al., 2007) cording to standard protocol (Mamontova et al., 2019). Stage-tips were prepared by filling of polypropylene Vertex pipette tips (200 mkl; SSIbio, USA) with six layers of C18 reversed phase excised from Empore 3M C18 extraction disks (3M-Corporation, Maplewood, MN, USA). The desalted peptides were evaporated in a Labconco Centrivap Centrifugal Concentrator (Labconco, USA) and dissolved in water/0.1% formic acid (LC-MS Grade; LiChrosolv) to approximate final concentration of 250 ng/mkl for further LC-MS/MS analysis. LC-MS/MS analysis. Approximate 500 ng of peptides were used for shotgun proteomics analysis by LC-MS/MS with ion mobility in TimsToF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) with nanoElute UHPLC system (Bruker Daltonics, Bremen, Germany). UHPLC was performed in one-column separation mode with Aurora Ultimate 25 cm separation column (C18 stationary phase, length 250 mm, diameter 0.075 mm, particle size 1.7 mkm, pore size 120 A; IonOpticks, Fitzroy, Australia) in gradient mode with 300 nL/min flow rate with column temperature at 60C. Phase A was water/0.1% formic acid, phase B was acetonitrile/0.1% formic acid (LC-MS Grade; LiChrosolv). The gradient was from 2% to 18% phase B for 44 min, then to 25% of phase B for 11 min, then to 37% of phase B for 5 min, then to 85% of phase B for 2 min with subsequent wash with 85% phase B for 15 min. The column was equilibrated with 4 column volumes before each sample. Parameters of ion source for electrospray ionization: 4500 V of capillary voltage, 3 l/min N2 flow, and 180C source temperature. The mass spectrometry acquisition was performed in DDA PASEF mode in positive polarity with the fragmentation of ions with at least two charges in m/z range from 100 to 1700 and ion mobility range from 0.60 to 1.60 1/K0. DDA-PASEF raw data files were analyzed in Peaks Xpro software (v.10.6) (Bioinformatics Solutions Inc., Canada) using human protein SwissProt database (uploaded on 19 January 2024). The search parameters are as follows: trypsin/P protease; 2 possible missed cleavage sites; maximum number of variable modifications on a peptide was set to 2; 13C(6) 15N(4) SILAC label, 13C(6) 15N(2) SILAC label, methionine oxidation, N-term acetylation, NQ-deamidation, carbamidomethylation on cysteine were possible modifications; precursor charge range from 2 to 7; parent mass error tolerance 10.0 ppm, fragment mass error tolerance 0.05 Da; FDR 1%.