Top-Down Individual Ion Mass Spectrometry Reveals 85-110 kDa Catenin Phospho-Proteoforms Regulated by Actomyosin Contractility

A central challenge in top-down proteomics is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I ² MS) for intact mass and fragmentation analysis of β- and α-catenins (85-110 kDa), key components of adherens junctions. Using denatured I ² MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on β-catenin and 7 on α-catenin, with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α-catenin S641 was constitutive, while other sites in the P-linker and actin-binding domains as well as β-catenin S675 and S552 were sensitive to actomyosin perturbation. Application of I ² MS for fragment ion detection (I ² MS ² ) also enabled 25-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry. Our results support a “catenin phospho-code” model, wherein combinatorial phosphorylation patterns encode mechano-transductive signals regulating cell–cell adhesion. This work establishes top-down I ² MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.