Online Cross-Linking of Peptides and Proteins during Contained-Electrospray Ionization Mass Spectrometry

enable all levels of protein structures to be characterized, including primary protein sequence, post-translational modifications, and threestudies by MS require the use of many separate techniques that are performed independently of each other. Herein, we described a contained-electrospray (ES) experiment that has potential to integrate peptide/protein cross-linking with the general MS workflow. In our experiment, cross-linking of protein/peptide occurs simultaneously with ionization after analytes, and cross-linkers are sprayed from two separate ES emitters. The online cross-linking process occurring in the charged microdroplet environment was optimized using trilysine peptide and bis(sulfosuccinimidyl)suberate cross-linker. We detected the electrostatic complex between analyte and cross-linker, the monolinked intermediate, and the fully cross-linked product, allowing us to correctly predict the sequence of reaction events in the crosslinking process. Importantly, we observed that the terminal fully cross-linked product is composed of two distinct conformations. In one form, the product involved cross-linking between two epsilon-NH2 amines in lysine residues, while the other conformer was formed by a reaction between one epsilon-NH2 amine and the N-terminus. The experimental conditions for selecting one cross-linked species over others during the online ES ionization-MS analysis have been detailed. Appropriate parameters enabled the reaction between alpha lactalbumin proteins and cross-linkers using a non-denaturing spray condition. These results establish a framework for a future development in high-throughput structural MS method, where all levels of protein information can be gathered in a single experiment.