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 (I²MS) for intact mass and fragmentation analysis of β- and α-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 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 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 (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 I²MS 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.