High-throughput quantitative proteomics enabled by mass defect-based 12-plex dileu isobaric tags

Abstract

Isobaric labeling has become a popular technique for high-throughput, mass spectrometry (MS)-based relative quantification of peptides and proteins. However, widespread use of the approach for large-scale proteomics applications has been limited by the high cost of commercial isobaric tags. To address this, we have developed our own N, N -dimethyl leucine (DiLeu) multiplex isobaric tags as a cost-effective alternative that can be synthesized with ease using readily available isotopic reagents. When paired with high- resolution tandem mass (MSn) acquisition, mass defect-based DiLeu isobaric tags allow relative quantification of up to twelve samples in a single liquid chromatography (LC)–MS2 experiment. Herein, we present detailed methods for synthesis of 12-plex DiLeu isobaric tags, labeling of complex protein digest samples, analysis by high-resolution nanoLC–MSn, and processing of acquired data.