RESUMO
BACKGROUND: The comprehensive analysis of human kidney glomerulus we previously performed using highly purified glomeruli, provided a dataset of 6,686 unique proteins representing 2,966 distinct genes. This dataset, however, contained considerable redundancy resulting from identification criteria under which all the proteins matched with the same set of peptides and its subset were reported as identified proteins. In this study we reanalyzed the raw data using the Mascot search engine and highly stringent criteria in order to select proteins with the highest scores matching peptides with scores exceeding the "Identity Threshold" and one or more unique peptides. This enabled us to exclude proteins with lower scores which only matched the same set of peptides or its subset. This approach provided a high-confidence, non-redundant dataset of identified proteins for extensive profiling, annotation, and comparison with other proteome datasets that can provide biologically relevant knowledge of glomerulus proteome. RESULTS: Protein identification using the Mascot search engine under highly stringent, computational strategy generated a non-redundant dataset of 1,817 proteins representing 1,478 genes. These proteins were represented by 2-D protein array specifying observed molecular weight and isoelectric point range of identified proteins to demonstrate differences in the observed and calculated physicochemical properties. Characteristics of glomerulus proteome could be illustrated by GO analysis and protein classification. The depth of proteomic analysis was well documented via comparison of the dynamic range of identified proteins with other proteomic analyses of human glomerulus, as well as a high coverage of biologically important pathways. Comparison of glomerulus proteome with human plasma and urine proteomes, provided by comprehensive analysis, suggested the extent and characteristics of proteins contaminated from plasma and excreted into urine, respectively. Among the latter proteins, several were demonstrated to be highly or specifically localized in the glomerulus by cross-reference analysis with the Human Protein Atlas database, and could be biomarker candidates for glomerular injury. Furthermore, comparison of ortholog proteins identified in human and mouse glomeruli suggest some biologically significant differences in glomerulus proteomes between the two species. CONCLUSIONS: A high-confidence, non-redundant dataset of proteins created by comprehensive proteomic analysis could provide a more extensive understanding of human glomerulus proteome and could be useful as a resource for the discovery of biomarkers and disease-relevant proteins.
RESUMO
PURPOSE: Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples. EXPERIMENTAL DESIGN: Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins. RESULTS: Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins. CONCLUSIONS AND CLINICAL RELEVANCE: Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study.