Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

Establishing a decision tree for native mass spectrometry analysis of membrane proteins in complex membrane mimetics (#191)

Weijing Liu 1 , Christopher Mullen 1 , Amanda E Lee 1 , Donggyun Kim 2 , Vadim Cherezov 2 , Gregory J Dodge 3 , Barbara Imperiali 3 , Hiruni S Jayasekera 4 , Michael Marty 4 , Rosa Viner 1
  1. Thermo Fisher Scientific, San Jose, CA, USA
  2. Bridge Institute, University of Southern California, Los Angelos, CA, USA
  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
  4. University of Arizona, Tucson, AZ, USA

Membrane proteins (MPs) represents 60% clinical drug targets owing to their active involvement in cellular processes. The complexity of membrane mimetics for MPs solubilization pose challenges for native mass spectrometry (nMS) analyses. Common membrane mimetics including detergent micelles, nanodiscs and styrene maleic-acid lipid particles (SMALPs) are critical for preserving native structure of MPs. Here, we aim to develop a decision tree for nMS characterization of MPs in different membrane mimetics on two mass spec based platforms, the Orbitrap Ascend Tribrid MS and the QE UHMR. Firstly, online buffer exchange-nMS (OBE-nMS) enables quick assessment of MPs in either detergent or nanodiscs. Secondly, either charge detection mass spectrometry on the QE UHMR or proton transfer charge reduction (PTCR) on the Orbitrap Ascend is compelling to resolve MPs in SMALPs. A decision tree approach was tested using a variety of MPs and demonstrates its complementarity to other structural biology tools. With this approach, we were able to detect 4 out of 5 different subunits plus PTMs on GPCR-Gs complex (>200 kDa) within 6 minutes.  Batch-to-batch variation screened out by OBE-nMS and Direct Mass Technology was later confirmed by Cryo-EM images. Additionally, the oligomerization is controversial of Wbap in SMALP. Direct Mass Technology results show its MW ~146 kDa in AmAc containing detergent and ~243kDa in AmAc only. The later MW reflects the intact dimeric structure corresponding to the mass photometry results. Lastly, proton transfer charge reduction helps resolve MPs in complex mimetics by isolation of a small subset of the m/z spectrum and then charge reducing it to reveal spatially resolved MPs. The resolved MPs in mimetics allow for the study of lipid composition and enable the potential for further MSn studies to complete characterization of the complex. In this work, we aim for a decision tree on native MS method selection for assessing MPs in different membrane mimetics for efficiently solving biological problems.