His-Tagged Peptidiscs Enable Affinity Purification of the Membrane Proteome for Downstream Mass Spectrometry Analysis
This week we profile a recent publication in the Journal of Proteome Research from Dr. John Young (pictured)
in the laboratory of Dr. Franck Duong in the Department of Biochemistry and Molecular Biology at UBC.
Can you provide a brief overview of your lab’s current research focus?
Our laboratory recently introduced the peptidisc membrane mimetic as a new tool for characterizing membrane proteins in detergent-free aqueous solution (https://elifesciences.org/articles/46615). The peptidisc enables rapid, streamlined reconstitution of the global E. coli membrane proteome into water-soluble particles after a brief detergent solubilization step. The reconstituted membrane protein library – or “peptidisc library” – is amenable to downstream analyses, including biochemical fractionation and/or mass spectrometry.
A major focus of our current work is how to further optimize the peptidisc library method. One outstanding problem is how to effectively enrich the low-abundance membrane proteome while removing high abundance cytosolic components such as ribosomes and other large soluble proteins from membrane preparations. During mass spectrometry analysis, the abundance of these contaminants masks detection of peptides from less abundant membrane proteins.
What is the significance of the findings in this publication?
We employ His-tagged peptidiscs to enrich the reconstituted membrane proteome by affinity chromatography. Using mass spectrometry, we show that the purified library sample is enriched in membrane proteins compared with the unpurified sample, and that ribosomal and other abundant soluble contaminants are strongly depleted. We apply this method to survey global changes in the membrane proteome upon depletion of the SecDFyajC complex, an important ancillary subunit of the universally conserved Sec translocon. In this depleted strain, we detect increased membrane localization of the motor ATPase SecA, along with increased levels of an unannotated inner membrane protein, YibN. These results demonstrate the utility of the peptidisc for global purification of membrane proteins and for monitoring changes in the membrane proteome.
What are the next steps for this research?
We were surprised and excited to observe that levels of the protein YibN increase upon depletion of SecDFyajC. What is the biological role of YibN? Our latest findings suggest that YibN may be involved in mediating Sec-dependent protein translocation and/or membrane protein insertion, possibly by modulating the activity of SecDFyajC. Our next objective is to verify the interaction of YibN with the Sec translocon, possibly through targeted affinity purification/mass spectrometry (AP/MS) experiments. We will also throughly probe the biological activity of YibN using a combination of genetic and biochemical methods.
This work was funded by:
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.D. The mass spectrometry infrastructure was supported by the Canada Foundation for Innovation, and methods by the BC Knowledge Development Fund and the Genome Canada/Genome BC through the Pan-Canadian Proteomics Centre (214PRO) to L.J.F.