Itraq Proteomic Profile of Mscs from Different Sublocalizations in Osteoarthritis and Healthy Donors

Purpose: Osteoarthritis (OA) is a joint disease characterized by joint homeostatic alterations leading to progressive cartilage degradation, subchondral bone sclerosis and joint destruction. Maintenance and restoration of joint cartilage and bone structures depend on chondrocytes and osteblasts, both arising from common progenitor mesenchymal stem cells (MSCs). Thus, MSCs have become interesting players in the field of regenerative medicine both to replace injured tissues or to promote tissue repair. However, molecular mechanisms governing their properties in OA remain largely unknown. Similarly, it is also unknown whether the biological niche of MSCs determines their different potential. In order to better understand the role of MSCs in health and disease, we aimed to describe, by quantitative proteomic analysis, the protein expression profile of MSCs in different sublocalizations in OA and non-OA subjects. Methods: MSCs were obtained from subchondral bone (SB) cartilage (C) and bone marrow(BM) sublocalizations of a hip OA patient (CX) and a control donor (subcapital fracture) (SF). Cells were isolated, cultured and expanded.Total protein content was isolated dried in air and then re-suspended in 25 μl Dissolution Buffer. Protein concentrations were determined by Bradford assay (Sigma-Aldrich, St. Louis, MO, USA). Equal amount of total protein for each condition were subjected to in-solution digestion, followed by iTRAQ labelling according to manufacturer instructions (ABSciex, Foster city, CA, USA). The samples were labelled as follows: CX-SB(113); CX-C(114); CX-BM(115) and SF-SB(116), SF-C(117); SF-BM(118). Two samples were additionally labelled CX-BM(119) and SF-BM(119) as internal contol. The samples were then mixed together and desalted with home-made C-18 Stage-tips (Rappsilber and Mann, 2003). Reverse Phase peptide separation was made in a LC system (Agilent Technologies, Santa Clara, CA, USA). Fractions were again separated in a nanoLC system (Tempo, Eksigent) automatically deposited on a MALDI plate and analyzed by MSMS in a 4800 MALDI-TOF/TOF system (ABSciex). Relative quantitative analysis was done using ProteinPilot software (ABSciex) that uses the Paragon™ Algorithm for protein identification and quantification. Only proteins identified with at least 95% confidence, a Prot Score (protein confidence measure) of at least 1.3, a p-value ≤ 0.05 and ratio (≠1) were considered as modulated. Ontological analysis to determine biological annotations or combinations of annotations significantly associated to the list of modulated proteins were analyzed with GeneCodis3 http://genecodis.cnb.csic.es). Results: A total of 828 proteins were identified with high confidence, 153 of them with p values >0.05. 9 out of 153 proteins were common to SB-MSCs, C-MSCs and BM-MSCs while 38, 25 and 27 respectively were unique to these sublocalizations. Comparison between OA and non-OA subjects revealed the upregulation of 67 proteins and the downregulation of 86 proteins in OA-MSCs. (Figure) Ontological classification of these proteins revealed the enrichment in cytoplasmic and cytosolic proteins. OA upregulated proteins mainly corresponding to: carbohydrate metabolism and aging biological processes, were enriched BM-MSCs. Cell proliferation, carbohydrate metabolism, signal transduction and protein folding in SB-MSCs; and Glycolysis/Gluconeogenesis and Protein processing in cartilage MSCs. Downregulated proteins were related with carbohydrate metabolism and cell death in SB and translation, protein folding, developmental maturation in Cartilage-MSCs. Conclusions: This study generates an initial proteome reference map of MSCs from OA and non-OA of three different sublocalizations, which will be valuable to functionally validate the role of differentially regulated proteins to better understand the underlying mechanism of MSCs differentiation.