Development of High-Sensitivity Quantitative Phosphoproteomics Using Motif-Targeting and Isobaric Tag-based Boosting Approaches

Abstract

Protein phosphorylation is a crucial regulatory mechanism in cellular signaling, yet comprehensive analysis of the phosphoproteome is challenging due to its extensive dynamic range and complexity. This review explores the chromatographic behavior of phosphopeptides, and the strategies developed to tackle the difficulties in detecting the low-abundance phosphoproteome, particularly focusing on motif-targeting phosphoproteomics that utilize protein kinase substrate specificity. The review highlights how in vitro kinase reactions facilitate the selective enrichment of specific subsets of the phosphoproteome, thereby reducing sample complexity and improving the detection of low-abundance species, such as tyrosine phosphorylation. Two complementary approaches are discussed: motif-targeting phosphoproteomics, which involves performing kinase reactions on target samples to create enriched phosphopeptide populations for direct analysis, and motif-centric phosphoproteomics, where kinase-generated phosphopeptides are isobarically labeled and used as carrier channels to enhance signals from low-abundance species. Additionally, recent advancements in phosphopeptide chromatography, including mobile phase optimization and temperature-dependent retention behavior, are examined. Applications in tyrosine phosphoproteome analysis and kinase inhibitor profiling illustrate the effectiveness of integrating biochemical selectivity with chromatographic separation for pathway-focused phosphoproteomics, even from limited sample amounts.