Development of Electrostatic-to-Covalent Gas Phase Cross-linkers for Protein Structure Measurements by Mass Spectrometry

The benefits of native mass spectrometry have led to the extensive study of proteins inside mass spectrometers in the gas phase. The expansion of native mass spectrometry requires novel tools for gaining greater insights into protein structures. Herein, we introduce a new approach utilizing gas phase ion/ion reactions, where cross-linking reagents link unprotonated lysine residues, arginine residues, and N-termini with their protonated forms. We used three lengths of linkers, determining that different length cross-linkers resulted in different residues being cross-linked, as we have previously observed for electrostatic-to-electrostatic cross-linkers. However, this new method allows for the probing of both protonated and neutral lysine and arginine residues. Native mass spectrometry often produces fewer charges than protonatable sites, allowing access to a greater number of sites on proteins using an electrostatic-to-covalent cross-linking approach. In this report, we describe the reaction phenomenology and trends at reaction sites. We envision electrostatic-to-covalent cross-linking as a useful structural tool to provide complementary information to other native MS-based measurements such as collision cross section.