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  • Mass spectrometry MS based proteomics

    2018-10-24

    Mass spectrometry (MS)-based proteomics is a powerful approach for the comparative analysis of protein pikfyve inhibitor between cell populations. Global approaches have been used to define the MSC proteome (Delorme et al., 2008; Mareddy et al., 2009; Mindaye et al., 2013a, 2013b) and to track the changes in membrane protein expression upon differentiation (Foster et al., 2005). However, comprehensive identification of specific surface markers has been limited by a lack of enrichment of membrane proteins, insufficient resolution of peptides prior to MS, and inability to compare protein levels between progenitors and differentiated cells.
    Results
    Discussion Our identification of proteins, with at least a 10-fold improvement in quantification compared with previous published studies, is a major leap in defining the mesenchymal progenitor cell-matrix interface proteome. Other groups have identified similar overall protein numbers and some similar markers when studying MSC populations (Mindaye et al., 2013a, 2013b), but previous attempts at quantifying differences in marker protein levels between cell types have been limited. One study, which used isotypic cysteine labeling to compare bone marrow and umbilical cord vein mesenchymal progenitor cells, enabled the quantification of 545 proteins, but represented a global proteomic approach with no pre-fractionation of membrane proteins prior to LC-MS/MS (Miranda et al., 2012). Using iTRAQ to simultaneously deliver relative quantification of multiple samples has major advantages over other MS methods for analyzing the membrane proteome, which are often poorly resolved using gel-based methods and, in the case of 2D-PAGE MS comparisons, rely on visible differences in silver stain intensity prior to protein identification (Kim et al., 2012). We and others have shown that protein and mRNA levels are not directly comparable (Miranda et al., 2012; Unwin and Whetton, 2006). Although published transcriptomic analyses comparing bone marrow MSCs with HDFs have identified some similar markers to those identified in this study, e.g., enrichment in ADAM12, VCAM1, and ALPL (Ren et al., 2011), there were also marked variations, underlining the essential need to define protein levels. Although well beyond the remit of this study to define the full significance of the expression of all identified MSC markers, we conducted selected knockdowns to gain initial insights. Knockdown of CD9, ROR2, and EPHA2 resulted in dramatic alterations in MSC phenotype. Comparable siRNA analyses of HUCPVC donor cultures showed similar results (unpublished data). CD9 is a tetraspanin receptor that interacts with and activates β1 integrin adhesion receptors, altering integrin-dependent cell migration (Kotha et al., 2008). Although CD9 is widely reported to be expressed by mesenchymal progenitor cells from different tissues and to influence integrin signaling, investigations into its function in MSCs are limited. We found that depletion of CD9 alters MSC proliferation, shape, and migration and blocks focal adhesions, thereby modifying cell fate. ROR2 is a member of an orphan receptor tyrosine kinase family. Ror2-deficient mice are neonatal lethal due to severe skeletal and heart defects (Takeuchi et al., 2000). Mutations in Ror2 in humans cause skeletal dysplasias (Afzal and Jeffery, 2003). ROR2 has been proposed to regulate osteogenic differentiation of MSCs in vitro possibly by acting as a Wnt co-receptor (Yun et al., 2014). We have shown that depletion of ROR2 not only downregulates osteogenic markers (COL1A1, BGLAP, RUNX2) in multipotent growth conditions, but also dramatically changes cell shape and inhibits cell proliferation and migration, suggesting that ROR2 crosstalk with Wnts may regulate mesodermal development. EPHA2 is a member of the family of Eph receptor tyrosine kinases that influence development and is often deregulated in cancer cells. It binds membrane-bound ephrin-A family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling that can modulate expression of mesodermal genes. EPHA2 may negatively regulate cell-ECM adhesion and cell growth due to suppression of focal adhesion kinase (Miao et al., 2000), whereas others have shown that ephrin-A-EphA2 signaling promotes FAK- and p130cas-dependent cell adhesion and cytoskeletal assembly (Carter et al., 2002). In agreement with the latter study, downregulation of EPHA2 in MSCs reduced focal adhesion formation, altering cell shape, inhibiting migration, and downregulating mesodermal gene expression. Similarities in knockdown phenotypes of CD9 and EPHA2 suggest that these proteins may function in overlapping pathways to maintain MSC fate.