Developing next-generation mass spectrometric platforms for molecular cartography of the cellular membranes

Within the crowded environment of the cell membrane, membrane proteins dynamically interact with both soluble and membrane-associated partners, as well as surrounding lipids, to form transient protein complexes that are central to cellular function. Growing evidence indicates that these assemblies are regulated by both specific protein-lipid interactions and the broader biophysical properties of the membrane itself. Therefore, to understand how membrane protein assemblies drive cellular responses to external stimuli, it is essential to study their functional homo and heteromeric organization directly within the lipid bilayer context. The overarching goal of this MIRA is to address these challenges over three independent projects by developing next-generation mass spectrometry (MS)-based technologies to directly analyze membrane protein complexes and their lipid environment with precise molecular resolution and nanoscale spatial resolution. Project 1 will develop a native MS platform to capture heteromeric complexes of membrane and soluble proteins directly from tunable invitro single or bridged dual lipid bilayers. Using the neuronal SNARE assembly as a model, we will detect multi-protein complexes formed at membrane contact sites and interrogate how specific lipids modulate their architecture. This will establish a versatile platform to probe transbilayer signaling assemblies with tunable membrane properties. Project 2 will build an nMS pipeline for directly detecting membrane protein complexes from native cell membranes. By integrating native vesicle purification, tandem-affinity enrichment, and native top-down and complex up fragmentation strategies, we will resolve the composition and stoichiometry of physiological MP assemblies. We will further integrate our membrane active polymer-based native nanodisc platform as an alternative avenue. Project 3 will develop a lipidomics-based method to map the nanoscale lipid domains surrounding endogenously expressed membrane proteins. Using custom polymers and ultra-sensitive nanoLC-MS, we will quantify enriched lipid classes around specific organelle-resident membrane proteins to demonstrate how within the same organellar membrane proteins are distributed in different lipid domains. Together, these projects will yield a generalizable, modular, and highly sensitive technological toolkit to interrogate membrane protein-lipid assemblies directly within complex biological membranes. The unifying goal is to bridge the molecular resolution of structural biology with the native context of cell membranes. These platforms will broadly advance our ability to decode membrane-associated signaling mechanisms and their dysregulation in human health and disease—an outcome aligned with the MIRA mission of supporting innovative, high-impact, and sustainable research programs. Project Number: 1R35GM164438-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Kallol Gupta | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $304,970 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-M (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11337268