Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: exploration of proteome after removal of blood-derived proteins.

Overview

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.