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

Abstract

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.

Gas-Phase Unfolding Reveals Stability Shifts Associated with Substrate Binding in Modular Polyketide Synthases

“…Prior CIU studies have focused on both building a complete mechanistic picture of gas-phase protein unfolding and deploying CIU technology to detect subtle changes in protein structure. − Specifically, CIU has been used to differentiate biosimilar antibodies, assess the specificity of lipid binding within membrane protein complexes, and distinguish conformationally selective kinase inhibitor binding modes. ,,− Despite these many successes, CIU has been primarily used to analyze monomeric proteins less than 150 kDa. , Furthermore, the stability differences reported within protein CIU data, often reported as CIU50 values that report the acceleration voltage required to convert 50% of the preceding conformer into the following intermediate state under a given set of instrument conditions, typically report on significant structural changes within smaller protein systems, leading to relatively large CIU50 differences. Clearly, pushing CIU studies toward protein complexes composed of large, multidomain subunits faces many analytical challenges.…”

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses

“…Such collision induced unfolding (CIU) experiments are realized through the stepwise heating of isolated gas-phase protein ions, where IM-MS is employed to record the changes in protein structure engendered during the collisional heating process. CIU fingerprinting data can reveal disulfide variants within biotherapeutic proteins and detect subtle differences in protein stability associated with local structure changes too subtle to detect by IM separation alone. ,− Altogether, native MS, IM, and CIU anchor a set of useful technologies that populate a continually expanding toolbox of structural MS technologies that have dramatically enhanced our understanding of protein complex architecture and function.…”