Abstract
Controlling selective fragmentation at particular residues in the gas phase could greatly improve our ability to characterize intact proteins by mass spectrometry and reveal proteoforms crucial to human biology. However, the chemical homogeneity and size of proteins frustrates characterization because fragmentation inevitably leads to thousands of product ions and highly complex spectra that resist complete interpretation. Herein, we report a method for the selective, photochemical cleavage of whole proteins in the gas phase via the photolysis of alpha-peptidyl radicals. The activation process results in predictable and selective dissociation of the peptide bond N-terminal to the initiating radical. Alpha peptidyl radicals are readily introduced in a residue specific manner via the nitrosation of tryptophan, which enables an easily triggered side chain loss. This approach affords highly controlled fragmentation of intact proteins in the gas phase, producing results analogous to those obtained by tryptic digests in solution. The smaller fragment ions produced by selective dissociation are abundant and can be further analyzed to facilitate characterization by providing more detailed information about sequence and modification sites.