Combining DiLeu Isobaric Labeling and Label-free Approaches for Metabolite Quantification and Biomarker Discovery of Lower Urinary Tract Symptoms (LUTS)

摘要

Mass spectrometry-based relative quantification is a useful technique to study the dysregulation of metabolites in disease states, usually through label-free or chemical derivatization approaches. Lower urinary tract symptoms (LUTS) commonly afflict aging males, including a range of urinary irritative and obstructive symptoms. Understanding the molecular mechanism of the multiple and complex manifestations of LUTS is challenging because of the lack of adequate biomarkers. In this study, an accurate and efficient relative quantification method for primary and secondary amine-containing small molecules was established using our lab's custom synthesized isobaric labels, 4-plex DiLeu. Four-plex isobaric DiLeu reagents were originally synthesized in our lab to label the N-terminus of a peptide for quantification. For the first time, we labeled aminecontaining metabolites with 4-plex DiLeu, combining with LC-ESI/MS and CE-ESI/MS platforms. Like other commercial isobaric tags (eg. TMT and iTRAQ), Isobaric DiLeu reagents have balanced structures with isotopes incorporation. The same compound labeled with 4-plex isobaric DiLeu produces a single peak in the MS scan. Upon MS/MS fragmentation, relative quantification can be achieved by comparing the intensities of DiLeu reporter ions (m/z 115, 116, 117, 118). Combining untargeted label-free and DiLeu labeling quantification methods allowed the identification and validation of candidate metabolic biomarkers of LUTS in human urine.