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Surfaceome Capture by Multiplex biotinylation (SUCAM) allows enhanced identification of cell surface proteins by mass spectrometry

ABSTRACT: Comprehensive interrogation of the cell surface proteome is critical for identifying targets for immunotherapy. Although surfaceomic technologies have advanced, current approaches remain limited by sensitivity and functional group specificity, restricting analyses to samples with large cell numbers and potentially overlooking subsets of surface proteins. To overcome these limitations, we developed Surfaceome Capture by Multiplex (SUCAM) biotinylation, a combinatorial strategy that employs multiplexed biotin reagents to derivatize multiple functional groups on cell surface proteins. Following enrichment with streptavidin magnetic beads, proteins were identified and quantified by LC–MS/MS. SUCAM enabled enhanced detection of plasma membrane and cell surface proteins compared with single-reagent methods, achieving robust identification from as few as 1.2 million cells. Replicate analyses demonstrated high reproducibility (mean coefficient of variation, 17%). Application of SUCAM to a panel of leukaemia cell lines recovered canonical leukaemic surface antigens as well as proteins with previously uncharacterised roles in this malignancy. These findings establish SUCAM as a complementary surfaceomic strategy that broadens proteome coverage and facilitates the discovery of therapeutic targets in both haematological and solid tumours.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Ana Levi

LAB HEAD: Pedro R.Cutillas

PROVIDER: PXD068779 | Pride | 2026-05-01

REPOSITORIES: Pride

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Project description:The dynamic protein distribution within and across the plasma membrane is pivotal in regulating cell communications. Yet, methods facilitating rapid labeling, applicable across diverse cell types, and amenable to multiplexed live imaging are scarce. We discovered that live mammalian cell surface proteins can be labeled using N-hydroxysuccinimide (NHS)-ester-based amine crosslinking of fluorescent dyes. Using mouse DC2.4 dendritic cells, human Jurkat T cells, and mouse primary lymphoma T cells as model systems, we revealed the membrane topology and membrane-associated cell communications previously challenging to capture by fluorescence microscopy techniques. In DC2.4 cells, we observed transient membrane protein accumulation at cell-cell contacts and their bidirectional migration patterns guided by membrane fibers. We further visualized the involvement of membrane fibers in the transfer of membrane-derived particles and ovalbumin, as well as caveolin-dependent endocytosis and subsequent transport of membrane labels in late endosomes and lysosomes. In addition, we demonstrate caveolin-1 phosphorylation-dependent insulin receptor endocytosis in HEK 293T cells. Volumetric structured illumination microscopy revealed changes in the membrane topology of Jurkat T cells in response to the activating surface, generation of membrane-derived microvesicles, and phagocytosis of CD3/CD28 Dynabeads on the glass surface. Finally, we demonstrate that the membrane label remains stable in the in vivo environment and evidence of intercellular membrane protein transfer from splenocytes from the donor T cell lymphoma SNF5 mouse to the healthy splenocytes in the C57/BL6 mouse 24 and 48-h after cell transfer. The ultrahigh membrane labeling density, coupled with high multiplexity and rapid labeling, enables innovative fluorescence microscopy applications to dissect membrane and membrane-derived structures in intercellular and intracellular communication both in vitro and in vivo.

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Surfaceome Capture by Multiplex biotinylation (SUCAM) allows enhanced identification of cell surface proteins by mass spectrometry

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