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Abstract
Hydroxyl radical based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in a wide range of solution conditions. In hydroxyl radical protein footprinting with X-rays (XFP), hydroxyl radicals (•OH) generated by water radiolysis covalently label multiple amino acid (AA) side chains simultaneously. However, HRPF technologies faces challenges in achieving their full potential due to the broad (>103) dynamic range of AA’s reactivity to •OH and the difficulty to detect slightly modified residues, particularly in peptides with highly reactive residues like methionine-containing peptides and in peptides containing all low reactive residues. To overcome this limitation, we developed a synchrotron-based multiplex labeling chemistry that utilizes CF3 radicals (•CF3) produced from a trifluoromethylation (TFM) reagent under controlled and optimized •OH doses generated by X-rays. We optimized the dual •CF3/•OH chemistry in this TFM labeling approach using six model peptides and lysozyme, there-by extending the existing •OH labeling platform with simultaneous •CF3 labeling. This optimization led to a two-fold increase in labeled AAs in multiplex TFM labeling, primarily by labeling to a greater degree AAs with low •OH reactivity via the •CF3 channel, while moderate and highly •OH reactive AAs were labeled in both •CF3 and •OH channels. Importantly, the low reactivity of methionine to •CF3 enabled the detection and quantification of additional AAs labeled by •CF3 across methionine-containing pep-tides. Consistent with observations in model peptides and protein, we observed a balanced dual •CF3/•OH chemistry and more uniform labeling of residues in both •CF3/•OH channels optimizing protein footprinting. Furthermore, the solvent accessibility of lysozyme residues directly correlated with •CF3 labeling demonstrating that multiplex labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.
Supplementary materials
Title
SI file_Multiplex labeling of peptides and proteins
Description
Multiplex TFM labeling was optimized with six model peptides by varying TFM reagent concentration and keeping a constant absorbed •OH amount (Figure S1, S-2), multiplex TFM labeling was optimized with six model peptides by varying absorbed •OH amount (Figure S2, S-3), Venn diagram to compare lysozyme residues labeled in HRPF and non-optimized multiplex TFM labeling experiments (Figure S3, S-4), The comparison of labeling efficiency for non-optimized multiplex TFM labeling against HRPF for six model peptides (Table S1, S-5). The comparison of reactivity coverage for optimized multiplex TFM labeling against HRPF for lysozyme (Table S2, S-6).
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