Abstract
Protein phosphorylation creates functionally distinct proteoforms through complex modification cascades, yet capturing their temporal dynamics and combinatorial patterns remains a major analytical challenge. Here, we introduce a hybrid precision mass spectrometry (MS) strategy that integrates intact mass measurements for temporal tracking, bottom-up MS analysis for site-specific kinetics, and top-down MS sequencing for proteoform characterization to resolve phosphorylation dynamics within intact kinase complexes. Using AMP-activated protein kinase (AMPK) as a model system, we uncover coordinated autophosphorylation cascades exhibiting kinetic hierarchies, with α1-S496 showing the highest kinetic efficiency. Allosteric ADaM-site activation bypasses canonical α1-T183 phosphorylation, enabling autophosphorylation even in activation-deficient mutants. Top-down MS sequencing identifies the predominant β1 proteoform as S24/25+S108 double phosphorylation, a pattern linking extranuclear distribution with allosteric responsiveness. Phosphatase competition shows PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected. This integrated strategy uncovers the proteoform dynamics underlying AMPK activation and provides a broadly applicable framework for studying phosphorylation-based regulation in kinases.