1 research outputs found
A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies
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
ānative mass spectrometry (MS)ā, 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 assembliesīøi.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 assembliesīøinformation
that is key to successful integrative structural elucidation of biological
systems