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A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies

Author:
Olinares, Paul Dominic B., Dunn, Amelia D., Padovan, Júlio C., Fernandez-Martinez, Javier, Rout, Michael P., Chait, Brian T.
Source:
Analytical chemistry 2016 v.88 no.5 pp. 2799-2807
ISSN:
1520-6882
Subject:
mass spectrometry, protein synthesis, stoichiometry
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.
Agid:
5304519