Mass Spectrometry of Laser-Initiated Carbene Reactions for Protein Topographic Analysis

摘要

ABSTRACT: We report a protein labeling method usingnnonselective carbene reactions of sufficiently high efficiency tonpermit detection bymass spectrometricmethods. The approachnuses a diazirine-modified amino acid (L-2-amino-4,40n-azipenta-nnoic acid, “photoleucine”) as a label source, which is convertednto a highly reactive carbene by pulsed laser photolysis at 355 nm.nLabeling of standard proteins and peptides (CaM, Mb, M13)nwas achieved with yields up to 390-fold higher than previousnstudies using methylene. Carbene labeling is sensitive tonchanges in protein topography brought about by conforma-ntional change and ligand binding. The modification of apo-CaM was 45 ( 7% higher than that of holo-CaM. Modification of thenCaM-M13 complex reflected a 39 ( 1% reduction in labeling for bound holo-CaM relative to free holo-CaM. Labeling yield isnindependent of protein concentration over approximately 2 orders of magnitude but is weakly dependent on the presence of othernchromophores in a photon-limited apparatus. The current configuration required 2 min of irradiation for full reagent conversion;nhowever, it is shown that comparable yields can be achieved with a single high-energy laser pulse (>100mJ/pulse, <10 ns), offering anlabeling method with high temporal resolution. We suggest a mechanism of labeling governed by limited carbene diffusion and thenprotein surface activity of the diazirine precursor. This surface activity is speculated to return a measure of selectivity relative tonmethylene labeling, which ultimately may be tunable.