In-Cell Mass Spectrometry and Ultraviolet Photodissociation Navigates the Intracellular Protein Heterogeneity

Directly probing the heterogeneous conformations of intracellular proteins within their native cellular environment remains a significant challenge in mass spectrometry (MS). Here, we establish an in-cell MS and ultraviolet photodissociation (UVPD) strategy that directly ejects proteins from living cells into a mass spectrometer, followed by 193 nm UVPD for structural analysis. Applying this approach to calmodulin (CaM), we reveal that it adopts more extended conformations within living cells compared with purified samples in vitro, highlighting the unique influence of intracellular environments on protein folding. Furthermore, UVPD analysis of calcium ion (Ca²⁺)-binding variants of CaM unveils not only the conformational heterogeneity induced by multiple Ca²⁺ modulations but also reveals distinct preferences of Ca²⁺ binding sites across different conformations. This strategy provides a powerful tool for interrogating the structure-function relationships of intracellular protein variants with sophisticated metal ion binding, paving the way for a deeper understanding of protein conformations within their native cellular context.