Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.
Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.
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DOI：
10.1021/acs.analchem.6b05003
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发表时间：
2017-02-21
影响因子：
7.4
通讯作者：
Evers AS
中科院分区：
文献类型：
--
作者：
Budelier MM;Cheng WW;Bergdoll L;Chen ZW;Abramson J;Krishnan K;Qian M;Covey DF;Janetka JW;Evers AS
Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that depend on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs) where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a Fast Ligand Identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. FLI-tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. FLI-tag was coupled to an alkynecontaining neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of CID fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using FLI-tag and ETD we identified Glu73 as the site of photo-incorporation of our neurosteroid ligand in the IMP, mVDAC1, and top-down MS confirmed a single site of photolabeling.
