Proteomic patterns in glomerular research, a laser capture microdissection and liquid chromatography-tandem mass spectrometry approach

Abstract

Abstract Introduction: Molecular techniques have the potential to shed light on glomerular diseases that conventional renal pathology may be unable to reveal. The aim of this study was to investigate whether proteomic patterns of glomeruli obtained from kidney biopsies can differentiate between minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS) and control groups (CTR). Methods: 18 formalin-fixed, paraffin-embedded (FFPE) renal biopsies comprising three groups of samples (CTR=3, MCD=6, FSGS=9) were subjected to label-free quantitative mass spectrometry. Glomeruli were excised from FFPE renal biopsies by laser capture microdissection (LCM) and, to increase both yield and protein identifications, single-pot solid-phase-enhanced sample preparation (SP3) digest method was applied. The samples were analyzed by mass spectrometry based shotgun proteomics. Results: The proteome profiling resulted in the identification of a total of 723 proteins. Multivariate analysis provided several proteins important in the separation of the three groups. Pattern Hunter analysis revealed moderate and high correlation of proteins against CTR-FSGS-MCD or CTR-MCD-FSGS patterns. The most significant pathways involved were associated with nephrin family and cytoskeleton interactions, as well as laminin/extracellular matrix related proteins. Univariate analysis revealed 58 significant different proteins among the three groups. Signaling pathways of these proteins were also associated with nephrin family interactions and cytoskeleton organization. Conclusions: This study demonstrates that mass spectrometry-based shotgun proteomic analysis of LCM glomeruli yields reproducible and quantitative data capable of discriminating between different disease conditions. Differentially expressed proteins provide insights into pathogenesis of glomerular disease.