Synergistic potential of sulfated hyaluronic acid and dexamethasone
Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts as they were shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess to synergistic potentials are rare. In this study, we aim gain insights into the mode-of-action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles and the extracellular matrix, combining classical biochemical assays with mass spectrometry-based proteomics and further supported by novel bioinformatical computation.
Sample Processing Protocol
Primary hBMSC were SILAC labeled for 31 days and treated with sulfated hyaluronic acid, dexamethasone or both for 22 days. The matrix vesicles were collected from cell medium and the cells were harvested and lysed in 6 M urea, 2 M Thiourea containing buffer. The lysates gained from either cells or matrix vesicles were mixed in a 1:1:1-ratio (‘heavy’:’medium’:’light’) and fractionated in a 1D-SDS-PAGE. The gels were sliced into 8 fractions for both cell and MV samples and in-gel digestion and peptide extraction were conducted. The gained peptide samples were analyzed using a LTQ Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a NanoAcquity system (Waters GmbH, Eschborn, Germany). The peptides were desalted for 5 min in a 180 µm x 20 mm C18 column (Waters) by washing with 99% solvent A (99.9% water, 0.1% formic acid) and 1% solvent B (99.9% acetonitrile, 0.1% formic acid). The peptide separation was conducted in a 75 µm x 150 mm BEH130 C18 column (Waters) using a non-linear increasing gradient until 40% B for 140 min followed by washing with up to to 85% B (10 min) and re-equilibrating to 1% B (20 min). The peptides were electrosprayed by a nano-ESI source (IonMax, Thermo Scientific, Bremen, Germany) at a voltage of 1.7 kV. A full MS scan was conducted in the Orbitrap mass analyzer (R = 60,000 at m/z 400, AGC target value of 2 x 106 with a max. injection time of 500 ms). The ten most abundant peptides exceeding a threshold of 2,000 were selected for fragmentation (collision induced dissociation with normalized collision energy of 35%) and MS/MS scans in the linear ion trap (AGC target value of 8,000 with a max. injection time of 200 ms). A dynamic exclusion for CID-MS/MS scans was set to 120 s with a relative exclusion mass tolerance of 4 ppm and limiting the exclusion list to 500 entries. The MS/MS peak lists were generated using the Xcalibur software (version 2.1).
Data Processing Protocol
The peptide and protein identification as well as quantification was conducted using MaxQuant (version 184.108.40.206) and the integrated search engine Andromeda. For a combined analysis all MS/MS files were used as input at once regardless the cellular fraction. The peptide and protein identification was based on a target-decoy search against a concatenated human reference proteome set (retrieved from UniprotKB) also including all proteins in reverse sequence direction and known contaminants. Carbamidomethylation of C was set as fixed modification wheras oxidation of M and Acetylation of protein n-term were set as modified modifications. The precursor mass tolerance was set to 20 ppm for first search and 4.5 ppm for main search. The fragment ion tolerance was set to 0.5 Da. Peptide identification was restricted to a FDR < 0.05. Protein identification was based on at least 2 peptides with a minimum of 1 unique peptide per protein applying a FDR < 0.05. For protein quantification D4-labeled lysine and 13C6-labeled arginine was set as medium SILAC labels whereas 13C6, 15N2-labeled lysine and 13C6, 15N4-labeled arginine was set as heavy SILAC labels
Johannes R. Schmidt, Helmholtz Centre for Environmental Research - UFZ
Kristin Schubert, Helmholtz Centre for Environmental Research - UFZ Department for Molecular Systems Biology (MOLSYB) Functional genomics group Leipzig/Germany ( lab head )
Schmidt JR, Vogel S, Moeller S, Kalkhof S, Schubert K, von Bergen M, Hempel U. Sulfated hyaluronic acid and dexamethasone possess a synergistic potential in the differentiation of osteoblasts from human bone marrow stromal cells. J Cell Biochem. 2018 PubMed: 30485523