Abstract
BackgroundWe have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. However, it is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. Therefore, in this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database.ResultsOne microgram of human glomerular protein digest was analyzed each on five LC- combined mass spectrometers (LIT-TOF, LTQ-Orbitrap, Q-TOF, LIT and MALDI-TOF/TOF) yielding 139, 185, 94, 255 and 108 proteins respectively identified with strict criteria to ensure high confidence (> 99%) and low false discovery rate (FDR) (< 1%). An integrated profile of 332 distinct glomerular proteins was subsequently generated without discerned bias due to protein physicochemical properties (pI and MW), of which around 60% were detected commonly by more than two LC-MS/MS platforms. Comparative analysis with the comprehensive database demonstrated 14 proteins uniquely identified in this study and more than 70% of identified proteins in small datasets were concentrated to the top abundant 500 in the comprehensive database which consists of 2775 non-redundant proteins.ConclusionThis study showed representative human glomerulus proteomic profiles obtained from biopsies through analysis of comparable amounts of samples by different mass spectrometry. Our results implicated that high abundant proteins are more likely to be reproducibly identified in multiple mass spectrometers runs and different mass spectrometers. Furthermore, many podocyte essential proteins such as nephrin, podocin, podocalyxin and synaptopodin were also identified from the small samples in this study. Bioinformatic enrichment analysis results extended our understanding of the major glomerular proteins about their subcellular distributions and functions. The present study indicated that the proteins localized in certain cellular compartments, such as actin cytoskeleton, mitochondrial matrix, cell surface, basolateral plasma membrane, contractile fiber, proteinaceous extracellular matrix and adherens junction, represent high abundant glomerular proteins and these subcellular structures are also highly significantly over-represented in the glomerulus compared to the whole human background.
Highlights
The kidney glomerulus plays a pivotal role in plasma ultrafiltration and is the locus of kidney diseases which frequently progress to irreversible chronic renal failure
We conducted the preliminary experiment of four information based acquisition (IBA) runs for one sample analysis in order to assess the efficiency of every IBA run on both peptide and protein identification
Trypsin digests from normal human glomerular proteins were analyzed on five mass spectrometers and the obtained mass spectrometry (MS)/MS spectral raw data was processed by MASCOT search engine against IPI human database
Summary
The kidney glomerulus plays a pivotal role in plasma ultrafiltration and is the locus of kidney diseases which frequently progress to irreversible chronic renal failure. We have previously performed large-scale proteomic analysis of human glomerulus and constructed an in-depth database (http://www.hkupp.org) for understanding the pathophysiology of renal diseases [1]. We aimed to analyze human glomerular proteins with low quantities which are comparable to those obtained from biopsies using different mass spectrometers and compare these results with the comprehensive database. We have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. It is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. In this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database
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