Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins

摘要

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 are dependent 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. The FLI tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide ligand adducts. The FLI tag was coupled to an alkyne-containing 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 collision-induced dissociation (CID) fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using the FLI tag and ETD, we identified Glu73 as the site of photoincorporation of our neurosteroid ligand in the IMP, mouse voltage-dependent anion channel-1 (mVDAC1), and top-down MS confirmed a single site of photolabeling.