title: Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.
abstract: 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.
authors.affiliation: Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Melissa M
authors.identifier: 0000-0002-1779-5757
authors.initials: MM
authors.lastname: Budelier
authors.affiliation: Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Wayland W L
authors.initials: WW
authors.lastname: Cheng
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, University of California at Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bergdoll
authors.affiliation: Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|The Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Zi-Wei
authors.initials: ZW
authors.lastname: Chen
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, University of California at Los Angeles , Los Angeles, California 90095, United States.|The Institute for Stem Cell Biology and Regenerative Medicine (instem), National Centre for Biological Sciences-Tata Institute of Fundamental Research , Bangalore 560065, Karnataka India.
authors.forename: Jeff
authors.initials: J
authors.lastname: Abramson
authors.affiliation: Department of Developmental Biology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Kathiresan
authors.initials: K
authors.lastname: Krishnan
authors.affiliation: Department of Developmental Biology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Mingxing
authors.initials: M
authors.lastname: Qian
authors.affiliation: Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|The Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|Department of Developmental Biology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|Department of Psychiatry, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: James W
authors.initials: JW
authors.lastname: Janetka
authors.affiliation: Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|The Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis , St. Louis, Missouri 63110, United States.|Department of Developmental Biology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.
authors.forename: Alex S
authors.identifier: 0000-0002-0342-0575
authors.initials: AS
authors.lastname: Evers
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D057930:Click Chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007553:Isotope Labeling; D008024:Ligands; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000480:Alkynes; D008024:Ligands; D010455:Peptides; C494763:Vdac1 protein, mouse; D050995:Voltage-Dependent Anion Channel 1