title: Tag and Capture Flow Hydrogen Exchange Mass Spectrometry with a Fluorous-Immobilized Probe.
abstract: Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one's ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy for overcoming this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis. Here we illustrate a new solution involving fluorous conjugation of a retrievable probe. The appended fluorous tag allows for facile immobilization on a fluorous surface. When a target protein is passed over the immobilized probe molecule, it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange. The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif. Efficient capture is demonstrated, and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS. Protein captured from a crude bacterial cell lysate could also be deuterated without the need for separate purification steps before HX MS. The advantages and disadvantages of the method are discussed in light of miniaturization and automation.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Marcsisin
authors.forename: Cary
authors.initials: C
authors.lastname: Liptak
authors.affiliation: ‡Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Marineau
authors.affiliation: ‡Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, United States.
authors.forename: James E
authors.initials: JE
authors.lastname: Bradner
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D005466:Fluorocarbons / Q000737:chemistry*; D006845:Hydrocarbons, Fluorinated; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry; D015336:Molecular Probe Techniques; D011506:Proteins / Q000032:analysis*; D012996:Solutions
chemical_list: D005466:Fluorocarbons; D006845:Hydrocarbons, Fluorinated; D011506:Proteins; D012996:Solutions; D006859:Hydrogen