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Abstract
Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Due to the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a “one-pot” reaction for multiplex labeling of protein side chains. Towards this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois’ reagent activated by X-ray mediated water radiolysis, followed by three different mass spectrometry methods to compare the reactivity of CF3 and OH radical labeling for all 20 protein side chains. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical activated trifluoromethyl chemistry as “one-pot” reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.
Supplementary materials
Title
Multiplex chemical labeling of amino acids for protein footprinting structure assessment
Description
The comparison of modification rate between high and low X-ray flux, (Figure S1, S-2) comparison of modification rate between 100 µM and 25 µM amino acids (Figure S2, S-3), reactivity of 20 natural amino acids towards •CF3 and •OH modifications as analyzed by polar HILIC column (Figure S3, S-4), calculated photon flux, beam power, and •OH yield at the XFP beamline for several attenuator thicknesses (Table S1, S-5), comparison of CF3 and OH modification rates of 3 amino acids between low and high X-ray flux (Table S2, S-6), retention time along with CF3 and OH modifications of amino acids as detected via nonpolar reverse phase (C18) coupled mass spectrometry (Table S3, S-7, S-8) and retention time along with CF3 and OH modifications of amino acids as detected via polar HILIC column coupled mass spectrometry (Table S4, S-9).
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