Custom 4-Plex DiLeu Isobaric Labels Enable Relative Quantification of Urinary Proteins in Men with Lower Urinary...

The relative quantification of proteins using liquid chromatography mass spectrometry (LC-MS) has allowed researchers to compile lists of potential disease markers. These complex quantitative workflows often include isobaric labeling of enzymatically-produced peptides to analyze their relative abundances across multiple samples in a single LC-MS run. Recent efforts by our lab have provided scientists with cost-effective alternatives to expensive commercial labels. Although the quantitative performance of these dimethyl leucine (DiLeu) labels has been reported using known ratios of complex protein and peptide standards, their potential in large-scale proteomics studies using a clinically relevant system has never been investigated. Our work rectifies this oversight by implementing 4-plex DiLeu to quantify proteins in the urine of aging human males who suffer from lower urinary tract symptoms (LUTS). Protein abundances in 25 LUTS and 15 control patients were compared, revealing that of the 836 proteins quantified, 50 were found to be differentially expressed (>20% change) and statistically significant (p-value <0.05). Gene ontology (GO) analysis of the differentiated proteins showed that many were involved in inflammatory responses and implicated in fibrosis. While confirmation of individual protein abundance changes would be required to verify protein expression, this study represents the first report using the custom isobaric label, 4-plex DiLeu, to quantify protein abundances in a clinically relevant system.