title: A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.
abstract: The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called "native mass spectrometry (MS)", wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assemblies-i.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states, rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. Here, to address this challenge, we present a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assemblies-information that is key to successful integrative structural elucidation of biological systems.
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University , New York, New York 10065, United States.
authors: Paul Dominic B
authors: PD
authors: Olinares
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University , New York, New York 10065, United States.
authors: Amelia D
authors: AD
authors: Dunn
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University , New York, New York 10065, United States.
authors: Júlio C
authors: JC
authors: Padovan
authors: Laboratory of Cellular and Structural Biology, The Rockefeller University , New York, New York 10065, United States.
authors: Javier
authors: J
authors: Fernandez-Martinez
authors: Laboratory of Cellular and Structural Biology, The Rockefeller University , New York, New York 10065, United States.
authors: Michael P
authors: MP
authors: Rout
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University , New York, New York 10065, United States.
authors: Brian T
authors: BT
authors: Chait
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011506:Proteins