Native Mass Spectrometry Enables Direct Detection of Endogenous Protein Complexes from Crude Lysates of Cultured...

Native mass spectrometry (MS) enables the direct observation of non-covalent protein interactions while preserving the higher-order structures of protein complexes. We previously developed direct native MS approaches for analyzing overexpressed protein complexes from crude lysates and single human cells, retaining their interactions with small molecules and drugs. Here, we demonstrate that this strategy can be extended to endogenous protein complexes, which are present at substantially lower abundance than the overexpressed protein complexes analyzed in previous direct native MS studies, by directly analyzing crude lysates of cultured human HEK293T cells without prior purification or buffer exchange. Although crude human cell lysates contain salts and matrix compounds that typically cause peak broadening and signal suppression, we successfully observed the endogenous ~145-kDa glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tetramer directly from unpurified human cell lysates, including its NAD⁺-bound forms. These results demonstrate the feasibility of directly detecting an endogenous high-molecular-weight protein complex and its cofactor-binding states from crude human cultured cell lysates, providing a basis for extending this approach to other endogenous protein assemblies and future single-cell native MS analyses.