Collision-Induced Unfolding, Tandem MS, Bottom-up Proteomics, and Interactomics for Identification of Protein Complexes in Native Surface Mass Spectrometry

Endogenously occurring salts and nonvolatile matrix components in untreated biological surfaces can suppress protein ionization and promote adduct formation, challenging protein identification. Characterization of labile proteins within biological specimens is particularly demanding because additional purification or sample treatment steps can be time-intensive and can disrupt noncovalent interactions. It is demonstrated that the combined use of collision-induced unfolding, tandem mass spectrometry, and bottom-up proteomics improves protein characterization in native surface mass spectrometry (NSMS). This multiprong analysis is achieved by acquiring NSMS, MS/MS, ion mobility (IM), and bottom-up proteomics data from a single surface extracted sample. The validity of this multiprong approach was confirmed by the successful characterization of nine surface-deposited proteins, with molecular weights ranging from 8 to 147 kDa, in two separate mixtures. Bottom-up proteomics provided a list of proteins to match against observed proteins in NSMS and their detected subunits in tandem MS. The method was applied to characterize endogenous proteins from untreated chicken liver samples. The subcapsular liver sampling for NSMS analysis allowed for the detection of endogenous proteins with molecular weights of up to ∼220 kDa. Moreover, using IM-MS, collision cross sections and collision-induced unfolding pathways of enzymatic proteins and protein complexes of up to 145 kDa were obtained.