title: Hydrogen Exchange Mass Spectrometry of Proteins at Langmuir Monolayers.
abstract: Hydrogen exchange (HX) mass spectrometry (MS) is valuable for providing conformational information for proteins/peptides that are very difficult to analyze with other methods such as peripheral membrane proteins and peptides that interact with membranes. We developed a new type of HX MS measurement that integrates Langmuir monolayers. A lipid monolayer was generated, a peptide or protein associated with it, and then the monolayer-associated peptide or protein was exposed to deuterium. The deuterated species was recovered from the monolayer, digested, and deuterium incorporation monitored by MS. Test peptides showed that deuterium recovery in an optimized protocol was equivalent to deuterium recovery in conventional solution HX MS. The reproducibility of the measurements was high, despite the requirement of generating a new monolayer for each deuterium labeling time. We validated that known conformational changes in the presence of a monolayer/membrane could be observed with the peptide melittin and the myristoylated protein Arf-1. Results in an accompanying paper show that the method can reveal details of conformational changes in a protein (HIV-1 Nef), which adopts a different conformation, depending on whether or not it is able to insert into the lipid layer. Overall, the HX MS Langmuir monolayer method provided new and meaningful conformational information for proteins that associate with lipid layers. The combination of HX MS results with neutron or X-ray reflection of the same proteins in Langmuir monolayers can be more informative than the isolated use of either method.
authors.affiliation: †Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115-5000, United States.
authors.forename: Gregory F
authors.initials: GF
authors.lastname: Pirrone
authors.affiliation: ‡Bioenergy and Defense Technologies, Sandia National Laboratories, Albuquerque, New Mexico 87185, United States.
authors.forename: Briana C
authors.initials: BC
authors.lastname: Vernon
authors.affiliation: ‡Bioenergy and Defense Technologies, Sandia National Laboratories, Albuquerque, New Mexico 87185, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kent
authors.affiliation: †Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115-5000, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry; D008555:Melitten / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005229:Fatty Acids, Monounsaturated; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D008555:Melitten; D006859:Hydrogen; D020823:ADP-Ribosylation Factor 1; C054211:9-tetradecenoic acid