His-Tagged Peptidiscs Enable Affinity Purification of the Membrane Proteome for Downstream Mass Spectrometry Analysis

Characterization of the integral
membrane proteome by mass spectrometry
(MS) remains challenging due its high complexity and inherent insolubility.
In a typical experiment, the cellular membranes are isolated, the
proteins are solubilized and fractionated, and the detergent micelles
are removed before MS analysis. Detergents are not compatible with
mass spectrometry, however, and their removal from biological samples
often results in reduced protein identification. As an alternative
to detergents, we recently developed the peptidisc membrane mimetic,
which allows entrapment of the cell envelope proteome into water-soluble
particles, termed a “peptidisc library”. Here, we employ
a His-tagged version of the peptidisc peptide scaffold to enrich the
reconstituted membrane proteome by affinity chromatography. This purification
step reduces the sample complexity by depleting ribosomal and soluble
proteins that often cosediment with cellular membranes. As a result,
the peptidisc library is enriched in low-abundance membrane proteins.
We apply this method to survey changes in the membrane proteome upon
depletion of the SecDFyajC complex, the ancillary subunit of the Sec
translocon. In the depleted strain, we detect increased membrane localization
of the motor ATPase SecA, along with increased levels of an unannotated
inner membrane protein, YibN. Together, these results demonstrate
the utility of the peptidisc for global purification of membrane proteins
and for monitoring change in the membrane proteome.