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

Department of Chemistry, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
Analytical Chemistry (Impact Factor: 5.83). 03/2011; 83(8):2913-20. DOI: 10.1021/ac102655f
Source: PubMed

ABSTRACT We report a protein labeling method using nonselective carbene reactions of sufficiently high efficiency to permit detection by mass spectrometric methods. The approach uses a diazirine-modified amino acid (l-2-amino-4,4'-azipentanoic acid, "photoleucine") as a label source, which is converted to a highly reactive carbene by pulsed laser photolysis at 355 nm. Labeling of standard proteins and peptides (CaM, Mb, M13) was achieved with yields up to 390-fold higher than previous studies using methylene. Carbene labeling is sensitive to changes in protein topography brought about by conformational change and ligand binding. The modification of apo-CaM was 45 ± 7% higher than that of holo-CaM. Modification of the CaM-M13 complex reflected a 39 ± 1% reduction in labeling for bound holo-CaM relative to free holo-CaM. Labeling yield is independent of protein concentration over approximately 2 orders of magnitude but is weakly dependent on the presence of other chromophores in a photon-limited apparatus. The current configuration required 2 min of irradiation for full reagent conversion; however, it is shown that comparable yields can be achieved with a single high-energy laser pulse (>100 mJ/pulse, <10 ns), offering a labeling method with high temporal resolution. We suggest a mechanism of labeling governed by limited carbene diffusion and the protein surface activity of the diazirine precursor. This surface activity is speculated to return a measure of selectivity relative to methylene labeling, which ultimately may be tunable.

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May 21, 2014