Abstract
Recent advancements in mass spectrometry (MS) have revolutionized quantitative proteomics, with multiplex isotope labeling emerging as a key strategy for enhancing accuracy, precision, and throughput. This tutorial review offers a comprehensive overview of multiplex isotope labeling techniques, including precursor-based, mass defect-based, reporter ion-based, and hybrid labeling methods. It details their fundamental principles, advantages, and inherent limitations along with strategies to mitigate the limitation of ratio-distortion. This review will also cover the applications and latest progress in these labeling techniques across various domains, including cancer biomarker discovery, neuroproteomics, post-translational modification analysis, cross-linking MS, and single-cell proteomics. This Review aims to provide guidance for researchers on selecting appropriate methods for their specific goals while also highlighting the potential future directions in this rapidly evolving field.