Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize, and Purify a Detergent-Sensitive Membrane Protein Assembly

The peptidisc membrane mimetic enables
global reconstitution of
the bacterial membrane proteome into water-soluble detergent-free
particles, termed peptidisc libraries. We present here a method that
combines peptidisc libraries and chromosomal-level gene tagging technology
with affinity purification and mass spectrometry (AP/MS) to stabilize
and identify fragile membrane protein complexes that exist at native
expression levels. This method circumvents common artifacts caused
by bait protein overproduction and protein complex dissociation due
to lengthy exposure to detergents during protein isolation. Using
the Escherichia coli Sec system as
a case study, we identify an expanded version of the translocon, termed
the HMD complex, consisting of nine different integral membrane subunits.
This complex is stable in peptidiscs but dissociates in detergents.
Guided by this native-level proteomic information, we design and validate
a procedure that enables purification of the HMD complex with minimal
protein dissociation. These results highlight the utility of peptidiscs
and AP/MS to discover and stabilize fragile membrane protein assemblies.
Data are available via ProteomeXchange with identifier PXD032315.