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Pository for cell signaling molecules, in addition to serving a structural scaffolding role for cells [22]. Proteomics has provided insights into constituents of the ECM and their regulation (Table 1). One approach involves stable isotope labeling of amino acids in cell culture (SILAC), which enables proteins that are newly produced by cells to be differentiated from culture media supplemented with serum [23]. Studies analyzing the proteins released from cell lines have defined ECM constituents that are produced by tumor cells. Combined liquid chromatography-tandem MS (LC-MS/MS) has been used to analyze proteins released into conditioned media, surface proteins and whole-cell lysates of murine lung cancer cells that have undergone epithelialmesenchymal transition (EMT, a process that allows tumor cells with an epithelial phenotype to convert to mesenchymal cells). These analyses revealed increasedexpression of a number of ECM proteins that indicate that the tumor cells are shaping their microenvironment, including: fibronectin, which binds to integrin receptors; a member of the collagen family, collagen 6A1; and members of the laminin family of glycoproteins, laminins A5, B2, and C1 [24]. Runt-related transcription factor 2 (RUNX2) is positively associated with tumor progression in prostate cancer [25]. LC-MS/MS analysis of secreted proteins that are upregulated after RUNX2 overexpression in prostate cancer cells revealed increased expression of a number of proteins, including basement membrane components laminins A5 and B1 [25]. A shotgun proteomic analysis of metastatic melanoma cell-line secretomes also identified the matrix proteins fibronectin and extracellular matrix protein 1, as well as the matricellular proteins SPARC (secreted protein, acidic, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10956013 cysteine rich) and osteopontin [26]. Studies in which EMT was Capecitabine induced in Madin-Darby canine kidney cells by H-RAS demonstrated both extracellular remodeling and decreased expression of basement membrane proteins, as well as increased expression of fibronectin, biglycan and SPARC [27,28]. Lai et al. [29] performed an extensive analysis ofHanash and Schliekelman Genome Medicine 2014, 6:12 http://genomemedicine.com/content/6/2/Page 4 of(a)Sample preparation(b)Antibody arrayLabeling Reverse-phase protein array Gel-based fractionation Liquid fractionationProtein digestionPeptide separationMass spectrometry analysisPeptide mass analysisPeptide fragmentation analysisProteinAntibodyData analysisFigure 3 Methodologies for proteomic analysis. (a) Experimental workflow of common mass spectrometry (MS)-based proteomic approaches. Proteins from particular compartments or from whole-cell lysates may be separated using gel electrophoresis or chromatography. Individual fractions are subsequently digested, followed by MS of peptide mixtures. (b) Affinity-based proteomic analysis is generally applied to intact proteins. Antibodies with defined specificities may be arrayed on a glass slide or membrane followed by incubation with a lysate. Alternatively, with reverse phase protein arrays, lysates are spotted and incubated with individual antibodies that target a specific protein.changes in ECM proteins during the development of hepatocellular carcinoma. They observed upregulation of collagen type IV, VI, VII, X, XIV, XV, XVI, and XVII, of splice variants Col4a2, Col6a2, and Col6a3 and of nidogen 1, decorin, and perlecan, among other proteins, indicating the utility of ECM profiling for hepatocellular carcin.