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Mapping the nanoscale organization of the human cell surface proteome reveals uncharacterized functional associations and surface antigen clusters

ABSTRACT: The cell surface is a dynamic interface central to immune activation, cell-cell communication, and signal transduction. Despite its functional significance, the spatial organization of cell surface proteins remains poorly understood. High-resolution fluorescence microscopy and proximity labeling have advanced studies of surface protein arrangement, but a systematic analysis of the spatial organization of the complete surface proteome remains unexplored. In this study, we systematically mapped the surface proteome of human T-lymphocytes and B-lymphoblasts using proximity labeling of 85 antigens, identified from over 100 antibodies tested for binding to surface-exposed proteins. These experiments were coupled with an optimized data-independent acquisition (DIA) mass spectrometry workflow to generate a robust dataset. Unsupervised clustering of the resulting interactome revealed functional modules, including well-characterized complexes such as the T-cell receptor and HLA class I/II, alongside novel clusters. Notably, we identified mitochondrial proteins localized to the surface, including the transcription factor TFAM, suggesting previously unappreciated roles for mitochondrial proteins at the plasma membrane. A high-accuracy machine learning classifier predicted over 6,000 surface protein associations, highlighting functional associations such as IL10RBs role as a negative regulator of type I interferon signaling. Spatial modeling of the surface proteome provided insights into protein dispersion patterns, distinguishing widely distributed proteins, such as CD45, from localized antigens, such as CD226. Interestingly, protein distribution appeared independent of abundance, pointing to active mechanisms regulating surface organization. This work provides a comprehensive map of the human surfaceome, offering a resource for exploring the spatial and functional dynamics of the cell membrane proteome.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Brendan Floyd Carolyn Bertozzi

PROVIDER: MSV000096983 | MassIVE | Tue Jan 28 20:33:00 GMT 2025

SECONDARY ACCESSION(S): PXD060303

REPOSITORIES: MassIVE

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Mapping the nanoscale organization of the human cell surface proteome reveals uncharacterized functional associations and surface antigen clusters

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