Intact Mass Profiling Reveals Phospho-Proteoforms of the Catenins (85-110 kDa) Regulated by Actomyosin Contractility.

A central challenge in top-down proteomics (TDP) 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 (β-cat) and 7 on α-catenin (α-cat), with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α-cat S641 was constitutive, while other sites in the P-linker and actin-binding domains, as well as β-cat 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 (MS). Our results are consistent with a "catenin phospho‑code" model, wherein combinatorial phosphorylation patterns reflect and potentially modulate the mechanotransductive environment at cell-cell adhesions. This work establishes top-down I(2)MS as a viable approach for probing complex post-translational modification (PTM) landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.