Abstract
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 (I2MS) for intact mass and fragmentation analysis of α- and β-catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on α-catenin and 7 on β-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 I2MS for fragment ion detection (I2MS2) 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 I2MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.