Characterization of chondrogenic progenitor cells using mass spectrometry and multidimensional separation approaches

Abstract

Modern day mass spectrometry (MS) has evolved into a powerful analytical technique, central to the field of proteomics. Currently, the identification and quantification of proteins from different biological samples can be performed routinely and on a large-scale. Despite the availability of fast and accurate mass spectrometers, deciphering the complex proteome of an organism necessitates the use of various separation techniques prior to the mass spectrometric analysis. Each separation technique offers a set of advantages and disadvantages and has its own power and limitations. Evaluation of the performance of the different separation approaches prior to the mass spectrometric analysis is a crucial step in improving the analytical power of the technique. The main aim of this study is to perform a proteomic characterization of chondrogenic progenitor cells (CPCs) using mass spectrometry and multidimensional separation approaches. CPCs are progenitor cells of mesenchymal origin, characterized by high chondrogenic potential, that migrate into the degenerating cartilage in late stages of osteoarthritis (Koelling, Kruegel et al. 2009). The mobilization of these cells from the bone marrow can be seen as a regenerative attempt of the cartilage, a tissue with very low reparative capacity. While the migratory and differentiation potential of CPCs has been studied in detail, additional information regarding the phenotype and the cellular fate of CPCs is needed. Results from this work show that CPCs upregulate proteins associated with the pericellular matrix and downregulate mesenchymal stem cells markers, when grown in a three dimensional cell culture, indicating ongoing chondrogenesis. In addition, this study reports multiple examples of the identification and quantification of not only single protein species but of a number of variants of the same protein which have different abundance in differentiating and undifferentiated CPCs. Moreover, a number of secreted signaling factors were discovered. The characterization of these different aspects of the protein expression profile of CPCs was done using different multidimensional separation methods. While the analysis of chondrogenic differentiation of CPCs could be performed using single dimension separation at the protein and peptide level, the identification of multiple protein species and secreted signaling molecules demanded the application of methods with higher separation power such as two-dimensional electrophoresis and a novel three-dimensional separation approach combining protein separation by molecular weight and peptide separation by isoelectric focusing and reversed phase liquid chromatography prior to the mass spectrometric analysis. 2 Overall, this work provides a rich body of information about the protein expression profile of CPCs and its changes during chondrogenesis in addition to the application and critical evaluation of several multidimensional separation approaches for proteomic analysis.