A Dual Detergent Strategy to Capture a Bacterial Outer Membrane Proteome in Peptidiscs for Characterization by Mass Spectrometry and Binding Assays.

ABSTRACT

The outer membrane of Gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics, and therefore is a prime target for antimicrobial discovery. To facilitate the discovery efforts, a precise knowledge of the outer membrane proteome, and possible variations during pathogenesis, is important. Characterization of the bacterial outer membrane remain challenging, however, and low throughput, due to the high hydrophobicity and relatively low abundance of this cell compartment. Here we adapt our peptidisc-based method to selectively isolate the outer membrane proteome before analysis by mass spectrometry. Using a dual detergent membrane solubilization approach, followed by protein purification in peptidiscs, we capture over 70 outer membrane proteins, including 26 integral ß-barrels and 26 lipoproteins. Many of these proteins are present at high peptide intensities, indicative of a high abundance in the library sample. We further show that the isolated outer membrane proteome can be employed in downstream ligand-binding assays. This peptidisc library made of outer membrane proteins may therefore be useful to systematically survey other bacterial outer membrane proteomes, but also as a nanoparticle format able to support the discovery of next-generation antimicrobials. Data are available via ProteomeXchange identifier PXD036749.