title: Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli.
abstract: Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.
authors.affiliation: Division of Nephrology and Hypertension, University of Louisville School of Medicine, Louisville, Kentucky, USA.
authors.forename: Liliane
authors.initials: L
authors.lastname: Hobeika
authors.affiliation: Division of Nephrology and Hypertension, University of Louisville School of Medicine, Louisville, Kentucky, USA.
authors.forename: Michelle T
authors.initials: MT
authors.lastname: Barati
authors.affiliation: Division of Nephrology and Hypertension, University of Louisville School of Medicine, Louisville, Kentucky, USA; Robley Rex VAMC, Louisville, Kentucky, USA.
authors.forename: Dawn J
authors.initials: DJ
authors.lastname: Caster
authors.affiliation: Division of Nephrology and Hypertension, University of Louisville School of Medicine, Louisville, Kentucky, USA; Robley Rex VAMC, Louisville, Kentucky, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: McLeish
authors.affiliation: Division of Nephrology and Hypertension, University of Louisville School of Medicine, Louisville, Kentucky, USA. Electronic address: mlmerc02@exchange.louisville.edu.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Merchant
mesh_terms: D015415:Biomarkers / Q000032:analysis; D001706:Biopsy; D030562:Databases, Protein; D005109:Extracellular Matrix / Q000737:chemistry* / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000032:analysis*; D005240:Feasibility Studies; D005404:Fixatives; D005557:Formaldehyde; D005629:Frozen Sections; D050533:Glomerular Basement Membrane / Q000737:chemistry* / Q000473:pathology; D006801:Humans; D007674:Kidney Diseases / Q000175:diagnosis / Q000378:metabolism*; D061346:Laser Capture Microdissection; D013058:Mass Spectrometry; D016612:Paraffin Embedding; D011237:Predictive Value of Tests; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods
chemical_list: D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D005404:Fixatives; D005557:Formaldehyde
keywords: extracellular matrix; glomeruli; human; laser capture microdissection; mass spectrometry