ac9b03827_si_001.pdf (2.14 MB)
Subresidue-Resolution Footprinting of Ligand–Protein Interactions by Carbene Chemistry and Ion Mobility–Mass Spectrometry
journal contribution
posted on 2019-12-11, 15:11 authored by Gaoyuan Lu, Xiaowei Xu, Gongyu Li, Huiyong Sun, Nian Wang, Yinxue Zhu, Ning Wan, Yatao Shi, Guangji Wang, Lingjun Li, Haiping Hao, Hui YeThe knowledge of ligand–protein interactions is
essential
for understanding fundamental biological processes and for the rational
design of drugs that target
such processes. Carbene footprinting efficiently labels proteinaceous
residues and has been used with mass spectrometry (MS) to map ligand–protein
interactions. Nevertheless, previous footprinting studies are typically
performed at the residue level, and therefore, the resolution may
not be high enough to couple with conventional crystallography techniques.
Herein we developed a subresidue footprinting strategy based on the
discovery that carbene labeling produces subresidue peptide isomers
and the intensity changes of these isomers in response to ligand binding
can be exploited to delineate ligand–protein topography at
the subresidue level. The established workflow combines carbene footprinting,
extended liquid chromatographic separation, and ion mobility (IM)–MS
for efficient separation and identification of subresidue isomers.
Analysis of representative subresidue isomers located within the binding
cleft of lysozyme and those produced from an amyloid-β segment
have both uncovered structural information heretofore unavailable
by residue-level footprinting. Lastly, a “real-world”
application shows that the reactivity changes of subresidue isomers
at Phe399 can identify the interactive nuances between estrogen-related
receptor α, a potential drug target for cancer and metabolic
diseases, with its three ligands. These findings have significant
implications for drug design. Taken together, we envision the subresidue-level
resolution enabled by IM–MS-coupled carbene footprinting can
bridge the gap between structural MS and the more-established biophysical
tools and ultimately facilitate diverse applications for fundamental
research and pharmaceutical development.