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• W2566778847 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. 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. Extracellular matrix (ECM) is a 3-dimensional network of cross-linked secreted proteins that exists in 2 major forms, a form that surrounds cells as a structural scaffold and a specialized ECM that forms basement membranes. Genomic and proteomic analysis determined that mammalian ECM consists of ∼300 core proteins, including 43 collagen subunits, 35 proteoglycans, and ∼200 complex glycoproteins.1Bonnans C. Chou J. Werb Z. Remodelling the extracellular matrix in development and disease.Nat Rev Mol Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (2302) Google Scholar, 2Hynes R.O. Naba A. Overview of the matrisome–an inventory of extracellular matrix constituents and functions.Cold Spring Harb Perspect Biol. 2012; 4: a004903Crossref Scopus (684) Google Scholar In addition to the core Matrisome, >700 matrix-associated proteins have been identified, including secreted proteins, growth factors, cytokines, and proteins that regulate ECM organization and remodeling.3Naba A. Clauser K.R. Hoersch S. et al.The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.Mol Cell Proteomics. 2012; 11 (M111.014647)Crossref PubMed Scopus (647) Google Scholar, 4Hynes R.O. The extracellular matrix: not just pretty fibrils.Science. 2009; 326: 1216-1219Crossref PubMed Scopus (2301) Google Scholar, 5Mott J.D. Werb Z. Regulation of matrix biology by matrix metalloproteinases.Curr Opin Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (874) Google Scholar, 6Lu P. Takai K. Weaver V.M. et al.Extracellular matrix degradation and remodeling in development and disease.Cold Spring Harb Perspect Biol. 2011; 3Crossref PubMed Scopus (1356) Google Scholar ECM in different tissues contains a more limited number of proteins, as studies of ECM from lung and colon contained 146 and 106 matrix proteins, respectively, of which only 84 were common to both tissues.3Naba A. Clauser K.R. Hoersch S. et al.The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.Mol Cell Proteomics. 2012; 11 (M111.014647)Crossref PubMed Scopus (647) Google Scholar ECM undergoes continual remodeling by adherent cells through the release of degradative enzymes and production of new ECM.1Bonnans C. Chou J. Werb Z. Remodelling the extracellular matrix in development and disease.Nat Rev Mol Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (2302) Google Scholar, 6Lu P. Takai K. Weaver V.M. et al.Extracellular matrix degradation and remodeling in development and disease.Cold Spring Harb Perspect Biol. 2011; 3Crossref PubMed Scopus (1356) Google Scholar Cells interact with ECM through matrix component receptors, including integrins, discoidin domain receptor tyrosine kinases, syndecans, CD44, and dystroglycan.2Hynes R.O. Naba A. Overview of the matrisome–an inventory of extracellular matrix constituents and functions.Cold Spring Harb Perspect Biol. 2012; 4: a004903Crossref Scopus (684) Google Scholar In addition to receptors for specific matrix components, cells express receptors for matrix-associated cytokines and growth factors, for degradative fragments of matrix components, and that recognize mechanical stress. Interaction of those receptors with their ligands activates intracellular signal transduction pathways that regulate cell adhesion, migration, proliferation, differentiation, and survival.2Hynes R.O. Naba A. Overview of the matrisome–an inventory of extracellular matrix constituents and functions.Cold Spring Harb Perspect Biol. 2012; 4: a004903Crossref Scopus (684) Google Scholar, 7Naba A. Clauser K.R. Ding H. et al.The extracellular matrix: tools and insights for the “omics” era.Matrix Biol. 2016; 49: 10-24Crossref PubMed Scopus (511) Google Scholar The signal transduction pathways activated by the interaction of cells with ECM also regulate synthesis and secretion of ECM proteins. Thus, cells constantly remodel their surrounding ECM, whereas ECM regulates diverse cell functions. Aberrant ECM remodeling contributes to a number of diseases and produces fibrosis and organ failure. The glomerular ECM is generated, organized, and maintained by all 3 resident cell types, podocytes, mesangial cells, and endothelium.8Byron A. Randles M.J. Humphries J.D. et al.Glomerular cell cross-talk influences composition and assembly of extracellular matrix.J Am Soc Nephrol. 2014; 25: 953-966Crossref PubMed Scopus (78) Google Scholar Glomerular ECM functions as a structural matrix surrounding mesangial cells and providing a scaffold for glomerular capillaries.9Borza C.M. Pozzi A. The role of cell-extracellular matrix interactions in glomerular injury.Exp Cell Res. 2012; 318: 1001-1010Crossref PubMed Scopus (23) Google Scholar The glomerular basement membrane (GBM) is a specialized ECM generated by and separating podocytes and fenestrated vascular endothelial cells.10Scott R.P. Quaggin S.E. Review series: the cell biology of renal filtration.J Cell Biol. 2015; 209: 199-210Crossref PubMed Scopus (217) Google Scholar An increased accumulation of glomerular ECM occurs in a number of diseases, including diabetic nephropathy, IgA nephropathy, and focal segmental glomerulosclerosis. Disordered ECM remodeling leading to glomerulosclerosis is postulated to represent a final common pathway of glomerular injury. Kidneys obtained from animal models of human disease have been used to provide insight into disease-specific ECM alterations. Xu et al.11Xu B.J. Shyr Y. Liang X. et al.Proteomic patterns and prediction of glomerulosclerosis and its mechanisms.J Am Soc Nephrol. 2005; 16: 2967-2975Crossref PubMed Scopus (87) Google Scholar analyzed glomeruli isolated by laser capture microdissection (LCMD) from the 5/6 nephrectomy rat model of focal segmental glomerulosclerosis to show that the proteomic pattern of nonsclerotic glomeruli was more similar to sclerotic than normal glomeruli. They also identified thymosin β4 in endothelial cells of sclerotic and nonsclerotic glomeruli, but not in normal glomeruli. Thus, identification of the components of the glomerular ECM under normal and diseased conditions may provide insight into glomerular physiology and the pathophysiology of a number of glomerular diseases. Several studies applied proteomic approaches to define normal ECM composition using whole glomeruli isolated from human kidneys retrieved for transplantation12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar or by nephrectomy.13Yoshida Y. Miyazaki K. Kamiie J. et al.Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.Proteomics. 2005; 5: 1083-1096Crossref PubMed Scopus (64) Google Scholar, 14Cui Z. Yoshida Y. Xu B. et al.Profiling and annotation of human kidney glomerulus proteome.Proteome Sci. 2013; 11: 13Crossref PubMed Scopus (16) Google Scholar Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar identified 144 core ECM and ECM-associated proteins from intact glomeruli isolated from human donor kidneys unsuitable for transplantation. Identifying the composition of glomerular ECM from the limited tissue available from human biopsies has been hindered by the technical difficulties of separating ECM from cellular compartments and the limited amount of protein available for mass spectrometry (MS) analysis. The goal of the current study was to determine the feasibility of using glomerular sections obtained by laser capture microdissection of kidney biopsy specimens for proteomic analysis of the glomerular ECM. The tissue type (formalin-fixed paraffin-embedded [FFPE] and frozen [FR]), protein extraction technique, and number of glomerular sections isolated by LCMD were studied. Our study demonstrated the feasibility of a proteomic analysis of glomerular ECM composition from glomerular sections isolated from limited renal tissue and expanded the list of known glomerular ECM proteins. To compare protein identification from different tissue preparations and protein extraction techniques, we first examined the total number of proteins and peptides identified by MS from varying numbers of glomerular sections. Figure 1 shows the total number of tandem mass spectrometry (MS2) events and of proteins identified for different numbers of glomerular sections from each tissue preparation. For FR tissue, the number of MS2 events and identified proteins was optimal at 100 glomerular sections (Figure 1a). For FFPE tissue, the number of MS2 events was more variable with optimal values at 75 and 140 glomerular sections. The number of proteins identified was at a plateau between 60 and 130 glomerular sections, but increased at 140 sections (Figure 1b). Based on these findings, 100 glomerular sections were selected as the target for use in all subsequent studies. To determine the reproducibility of protein identification by MS analysis, peptides from all 3 protein extraction methods of both FFPE and FR tissue were analyzed twice by MS. Replicates 1 and 2 correspond to proteins identified from each of the 2 analyses. Comparison of the 1097 proteins in replicate 1 with the 1082 proteins in replicate 2 is shown in Figure 2 (see also Supplementary Table S1). A total of 906 proteins were identified in both replicates, whereas 191 proteins were found only in replicate 1 and 176 proteins were only identified in replicate 2. Thus, performing duplicate MS analyses of a single peptide mixture from extracted glomerular proteins increased the number of proteins identified by ∼20%. Because a goal of this project was ECM protein identification from limited kidney tissue, all proteins identified from replicates 1 and 2 were compared with the 1027 matrix proteins in the Matrisome database (http://matrisomeproject.mit.edu), a highly curated database of known ECM and ECM-associated proteins. A total of 112 matrix proteins were identified, of which 95 proteins were present in both replicates, 10 proteins were present only in replicate 1, and 7 proteins were present only in replicate 2 (Figure 2). To determine an effective method of protein extraction for matrix protein identification, 3 methods were examined: (i) extraction with ProteaseMAX (Promega, Inc., Madison, WI) Surfactant + heating (MAX), (ii) sequential extraction with 0.4% sodium dodecylsulfate (SDS) to remove cellular proteins followed by MAX (SDS), and (iii) sequential extraction with NH4OH/0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) (TX) to remove cellular proteins followed by MAX (TX). Sequential extraction with either SDS or TX, compared with MAX, resulted in the identification of a larger number of total proteins and proteins common to the Matrisome in both FFPE and FR tissue (Table 1). Matrix proteins obtained from FFPE tissue using MAX (N = 63), SDS (N = 81), and TX (N = 89) were compared. Of the matrix proteins identified by MAX, 94% were also identified by SDS and TX. More than 80% of the matrix proteins identified by sequential extraction were common to TX and SDS. Figure 3a shows that TX extraction identified a greater number of unique matrix proteins (N = 14) compared with SDS (N = 5) and MAX (N = 1). Figure 3b shows the comparison of matrix proteins identified from FR tissue via MAX (N = 66), SDS (N = 80), and TX (N = 79). Of the matrix proteins identified by MAX, 89% were also identified by SDS and TX. Of the matrix proteins identified by sequential extraction methods, 86% were common to TX and SDS, 9 proteins were unique to TX, 6 to SDS, and 1 to MAX. Our data indicate that sequential extraction in which cellular proteins are removed, particularly that using TX, provided optimal identification of matrix proteins.Table 1Comparison of the number of total and matrix proteins identified by MAX, SDS, and TX in both FFPE and FR tissueMAXSDSTXFFPE total574649689FR total694750874FFPE matrix638189FR matrix668079FFPE, formalin-fixed paraffin-embedded; FR, frozen; MAX, ProteaseMAX surfactant; SDS, sodium dodecylsulfate; TX, Triton X-100. Open table in a new tab FFPE, formalin-fixed paraffin-embedded; FR, frozen; MAX, ProteaseMAX surfactant; SDS, sodium dodecylsulfate; TX, Triton X-100. To determine the effect of tissue processing on matrix protein recovery, the matrix proteins identified in glomerular sections from FFPE tissue and FR tissue were compared for each extraction method. Of the matrix proteins identified by MAX, 55 were common between FFPE (87%) and FR (83%) tissue. Of the matrix proteins identified by sequential extraction with SDS, 66 were common between FFPE (81%) and FR (82%) tissue. Sequential extraction with TX identified 67 proteins common between FFPE (75%) and FR (85%) tissue. Our results suggest that identification of matrix proteins from FR tissue and FFPE preserved tissue is similar for each of the protein extraction methods. To determine the distribution of matrix proteins identified from cellular and decellularized fractions after sequential extraction with SDS and TX, matrix proteins identified in each fraction were compared. In glomerular sections from FFPE tissue, extraction with SDS resulted in identification of 70 matrix proteins in decellularized fractions and 31 matrix proteins in the cellular fraction, 20 proteins were common to both fractions (Figure 4a). Extraction with TX identified 63 matrix proteins in the decellularized fraction and 59 matrix proteins in the cellular fraction with 33 proteins common to both (Figure 4b). Both cellular and decellularized fractions contained glomerular structural and GBM proteins (Supplementary Table S2). In glomerular sections from FR tissue, sequential extraction with SDS identified 65 matrix proteins in the decellularized fraction and 63 matrix proteins in the cellular fraction with 48 proteins common to both (Figure 4c). For extraction by TX, 64 matrix proteins were present in the cellular fraction compared with 55 in the decellularized fraction with 40 matrix proteins in common (Figure 4d). This analysis indicates that MS analysis of both cellular and decellularized fractions provides the most complete identification of matrix proteins. To determine the validity of using a number of glomerular sections available from kidney biopsies for ECM protein identification, we combined proteins from all 3 extraction techniques of both FFPE and FR tissue and compared that dataset with the Matrisome dataset and with a previously published report that identified the largest number of ECM proteins using whole glomeruli isolated from 3 human donor kidneys unsuitable for transplantation.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar Our dataset contained 112 proteins present in the Matrisome project dataset, and we identified an additional 35 proteins previously reported by Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar but not contained in the Matrisome dataset. Thus, 147 glomerular ECM proteins were identified in the current study, of which 91 proteins were previously identified by Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar The current study identified 54 new glomerular ECM candidate proteins (Figure 5). Analysis of these 54 proteins using the Human Protein Atlas Database found that 22 proteins were previously shown to be present in glomeruli alone or in both glomeruli and tubules, 14 proteins were present in tubules only, and 18 proteins were not found in either glomeruli or tubules (Table 2).Table 2Localization of the newly identified ECM associated proteins in the kidney using the Human Protein Atlas DatabaseGene nameLocationA2MPresent in glomeruli and tubulesA2ML1Not detected in either tubules or glomeruliANGPTL6Not availableANXA1Present in glomeruli, not tubulesANXA3Present in glomeruli < tubulesANXA4Present in tubules, not detected in glomeruliANXA6Present in glomeruli < tubulesCOL5A1Not detected in either tubules or glomeruliCRELD1Present in tubules, not detected in glomeruliCRIM1Present in glomeruli, not tubulesCST6Present in tubules, not detected in glomeruliCSTANot detected in either tubules or glomeruliCSTBNot detected in either tubules or glomeruliCTGFPresent in glomeruli and tubulesCTSAPresent in tubules, not detected in glomeruliCTSBPresent in tubules, not detected in glomeruliCTSZPresent in tubules, not detected in glomeruliDMBT1Present in tubules, not detected in glomeruliECM1Not detected in either tubules or glomeruliFBN2Not detected in either tubules or glomeruliFGF1Present in glomeruli>tubulesFLGNot detected in either tubules or glomeruliHRGPresent in glomeruli<tubulesHRNRPresent in glomeruli<tubulesITIH5Present in glomeruli and tubulesLGALS7Present in tubules, not detected in glomeruliLUMPresent in tubules, not detected in glomeruliMFAP2Not detected in either tubules or glomeruliMMRN2Present in glomeruli and tubulesMUC5BNot detected in either tubules or glomeruliNTN4Present in glomeruli<tubulesPAPLNPresent in glomeruli and tubulesPLGPresent in glomeruli and tubulesPLXNB2Present in glomeruli<tubulesS100A10Present in glomeruli and tubulesS100A11Present in tubules, not detected in glomeruliS100A12Not detected in either tubules or glomeruliS100A14Present in tubules, not detected in glomeruliS100A7Not detected in either tubules or glomeruliS100A9Not detected in either tubules or glomeruliS100PNot detected in either tubules or glomeruliSBSPONPresent in glomeruli<tubulesSERPINB1Not detected in either tubules or glomeruliSERPINB12Present in tubules, not detected in glomeruliSERPINB3Not detected in either tubules or glomeruliSERPINB6Present in glomeruli and tubulesSERPINB9Present in glomeruli not in tubulesSLPIPresent in tubules, not detected in glomeruliSPARCPresent in glomeruli>tubulesTGM1Not detected in either tubules or glomeruliTGM3Present in tubules, not detected in glomeruliTHSD4Present in glomeruli<tubulesTNCPresent in glomeruli not in tubulesVWFNot detected in either tubules or glomeruliECM, extracellular matrix. Open table in a new tab ECM, extracellular matrix. Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar divided their 144 glomerular ECM proteins into structural (24 proteins), basement membrane (24 proteins), and matrix-associated (96) proteins. We compared our ECM proteins with the proteins in each of those 3 categories. Our dataset contained 17 of 24 structural matrix proteins (Table 3), 19 of 24 basement membrane proteins in addition to 2 new basement membrane proteins (LAMA3 and LAMA4) (Table 4), and 55 of 96 matrix-associated proteins (Figure 5).Table 3Comparison of the structural ECM proteins identified in this study to those identified by Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google ScholarStructural ECM proteins,Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar(N = 24)Current study(N = 17)ASPNBGNBGNCOL12A1COL1A1COL1A1COL1A2COL1A2COL3A1COL3A1COL6A1COL6A1COL6A2COL6A2COL6A3COL6A3DCNDPTEMILIN1EMILIN1FGAFGAFGBFGBFGGFGGMGPMGPNPNTNPNTPOSTNPOSTNRPESPTGFBITGFBITINAGL1TINAGL1VTNVTNVWA5B2VWA8ECM, extracellular matrix. Open table in a new tab Table 4Comparison of the GBM proteins identified in this study with those identified by Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google ScholarGBM proteins, Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar (N = 24)GBM proteins, current study (N = 21)AGRNAGRNCOL15A1COL18A1COL18A1COL4A1COL4A1COL4A2COL4A2COL4A3COL4A3COL4A4COL4A4COL4A5COL4A5COL4A6FBLN1FBLN1FBN1FBN1FN1FN1FRAS1HMCN1HSPG2HSPG2LAMA2LAMA2LAMA3LAMA4LAMA5LAMA5LAMB1LAMB1LAMB2LAMB2LAMC1LAMC1NID1NID1NID2NID2TINAGVWA1VWA1GBM, glomerular basement membrane. Open table in a new tab ECM, extracellular matrix. GBM, glomerular basement membrane. All glomerular ECM proteins identified in the current study were converted to a protein interaction network model using the Search Tool for the Retrieval of Interacting Genes (STRING v10) database with the highest confidence score (0.900).15Szklarczyk D. Franceschini A. Wyder S. et al.STRING v10: protein-protein interaction networks, integrated over the tree of life.Nucleic Acids Res. 2015; 43: D447-D452Crossref PubMed Scopus (6748) Google Scholar Seven clusters of interacting proteins were identified, including basement membrane and structural ECM proteins composed of 13 collagens, 6 laminins, and 8 heparan sulfate proteoglycan GBM proteins (Figure 6a). Other clusters included 5 complement components, 5 matrix remodeling enzymes with cathepsin B as the central node, a group of 15 proteins involved in matrix remodeling with plasminogen as a central node, and a group of 10 proteins centered on apolipoprotein A1. The same analysis was performed after combining ECM proteins identified in this study and the report by Lennon et al.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar (Figure 6b). In addition to adding proteins to the networks composed of proteins of the current study, 2 new networks were identified. The first contained 7 proteins, with matrix metalloproteinase 9 as the central node, that participate in cell-matrix interactions and cell migration. The second contained 6 proteins composed of enzymes, including angiotensinogen, kininogen, cathepsin G, and cathepsin Z. Glomerular ECM is histologically abnormal in a number of diseases, and the ability to delineate ECM composition using patient biopsy specimens could identify diagnostic and prognostic changes and define the molecular events leading to glomerular ECM remodeling. A limitation of all proteomic studies to date is the inability to differentially enrich basement membrane from other forms of ECM such as mesangial matrix. This current study capitalizes on the unusual physical properties of ECM that often create challenges for standard protein extraction techniques, such as the insoluble components of ECM that are frequently discarded during sample preparation. Furthermore, the presence of highly abundant cytoplasmic and mitochondrial proteins limits detection of lower abundance ECM proteins. The current study combined enrichment of glomerular ECM by LCMD and sequential protein extraction with highly sensitive MS to identify ECM components from a number of glomerular sections compatible with those available from renal biopsies. We identified a majority of the structural and basement membrane glomerular ECM proteins, and the total number of ECM proteins found was comparable to that previously reported using whole glomeruli isolated from 3 human kidneys.12Lennon R. Byron A. Humphries J.D. et al.Global analysis reveals the complexity of the human glomerular extracellular matrix.J Am Soc Nephrol. 2014; 25: 939-951Crossref PubMed Scopus (119) Google Scholar A number of studies have shown the feasibility of obtaining glomerular sections for proteomic studies using LCMD.16Satoskar A.A. Shapiro J.P. Bott C.N. et al.Characterization of glomerular diseases using proteomic analysis of laser capture microdissected glomeruli.Mod Pathol. 2012; 25: 709-721Crossref PubMed Scopus (44) Google Scholar, 17Yoshida Y. Nameta M. Kuwano M. et al.Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: exploration of proteome after removal of blood-derived proteins.Proteomics Clin Appl. 2012; 6: 412-417Crossref PubMed Scopus (6) Google Scholar The combination of LCMD and MS identified proteins responsible for glomerular amyloid deposits,18Sethi S. Vrana J.A. Theis J.D. et al.Laser microdissection and mass spectrometry-based proteomics aids the diagnosis and typing of renal amyloidosis.Kidney Int. 2012; 82: 226-234Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 19Sethi S. Theis J.D. Vrana J.A. et al.Laser microdissection and proteomic analysis of amyloidosis, cryoglobulinemic GN, fibrillary GN, and immunotactoid glomerulopathy.Clin J Am Soc Nephrol. 2013; 8: 915-921Crossref PubMed Scopus (67) Google Scholar defined targets of autoantibodies in glomerular immune complexes from membranous nephropathy and lupus nephritis patients,20Bruschi M. Carnevali M.L. Murtas C. et al.Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: alfa-enolase and borderline antigens.J Proteomics. 2011; 74: 2008-2017Crossref PubMed Scopus (102) Google Scholar, 21Bruschi M. Sinico R.A. Moroni G. et al.Glomerular autoimmune multicomponents of human lupus nephritis in vivo: alpha-enolase and annexin AI.J Am Soc Nephrol. 2014; 25: 2483-2498Crossref PubMed Scopus (81) Google Scholar, 22Bruschi M. Galetti M. Sinico R.A. et al.Glomerular autoimmune multicomponents of human lupus nephritis in vivo (2): planted antigens.J Am Soc Nephrol. 2015; 26: 1905-1924Crossref PubMed Scopus (45) Google Scholar identified a patient with IgD heavy-chain disease23Royal V. Q" @default.
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• W2566778847 title "Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli" @default.
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