Analysis of the extracellular matrix proteome by mass spectrometry Free

LB-306

The extracellular matrix (ECM) is comprised of various proteins that are not only important for cell survival, differentiation and cell-to-cell interaction, but also play critical roles in diseases such as osteoporosis, muscular dystrophy and tumor metastasis. To better understand the protein components in the ECM, we conducted a comprehensive proteomic analysis of the extracellular proteomes in fibroblast primary cultures derived from the wild-type and Lamin A deficiency mouse models. The Lamin A mutant mice exhibit severe retardation in postnatal development. The muscular dystrophy phenotype in the mutant mice, especially the abnormality in the ECM formation, resembles the same defect in human Emery-Dreifuss muscular dystrophy (EDMD). To elucidate the role these ECM proteins play in the muscular dystrophy, fibroblasts were isolated from both wild type and mutant mice. Then two protein extraction and enrichment methods, including lysing cells with deoxycholate or a short exposure of the cells to blendzyme, were applied to isolate ECM proteins from the fibroblasts. The enriched ECM proteins were further subjected to two approaches of proteomic analysis. The first approach was in-gel digestion, in which the ECM proteins were resolved on SDS-PAGE gel. The differential protein bands between the wild-type and mutant mice were excised, digested and analyzed by LC-MS/MS. Alternatively, the proteins were digested and the peptides were fractionated by strong ionic exchange HPLC. This was followed by the analysis of peptide fractions using LC-MS/MS. A total of 60 protein gel bands and more than 200 peptide fractions were analyzed. This resulted in the identification of more than 1,000 proteins in the sample prepared using deoxycholate and more than 300 proteins in the sample prepared using blendzyme. The combination of different protein extraction and sample preparation approaches improved the detection and coverage of ECM proteins. The comparison of the ECM proteomes in the Lamin A mutant versus mutant mice facilitated the search for proteins associated with the muscular dystrophy phenotype in the mouse models as well as the human EDMD. In summary, this study represents by far the most comprehensive analysis of the ECM proteome by mass spectrometry.