Flow cytometric analysis of HL60 cells LUX-labeled with increasing CG1-SOG concentrations showed light-induced and concentration-dependent cell-surface biotinylation (Supplementary Fig.?6). using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms Benzethonium Chloride and to discover surfaceome receptor nanoscale business with direct implications for drug action. Furthermore, by coupling SOG to Benzethonium Chloride antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+?T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies. test. Green and blue dots represent known and previously unknown CD20 associated proteins, respectively. Orange and red dots Rabbit Polyclonal to ACAD10 represent chains of the used antibody and the primary binding target CD20. Right, literature and LUX-MS-based cell surface conversation network of CD20. g Venn diagram showing overlap of CD20 proximal candidates identified Benzethonium Chloride on resting human B cells (Ramos) using horseradish peroxidase (HRP) conjugated antibodies or LUX-MS performed once in water (H2O) or heavy water (D2O) based buffer. h PCA analysis of CD38, CD54, CD166, and CD220 receptor microenvironments identified by antibody-guided LUX-MS on living B-lymphoma SUDHL6 cells. Source data are provided as a Source Data file and interactive volcano plots (Supplementary Data?1). We then examined LUX-labeling dynamics using Ab-SOG on a human B-lymphoma cell line and observed light-dependent cell surface biotinylation to be fine-tunable by the duration of illumination (Fig.?2c and Supplementary Fig.?2). The extend of labeling Benzethonium Chloride was further tunable by modulating buffer conditions, i.e., increasing D2O concentration and pH in line with enhanced photo-oxidation (Supplementary Fig.?4) and histidine conversion53, respectively. We then performed a CD20-targeted LUX-MS experiment by treating cultured human B-lymphoma cells with Ab-SOG prior illumination for 0C5?min. In total, 1674 proteins were quantified by LUX-MS with at least two unique proteotypic peptides per protein (Fig.?2d and Supplementary Fig.?2). Most proteins were equally abundant in the non-illuminated conditions, however, 63 proteins showed striking enrichment that correlated with the duration of illumination and that culminated in a fivefold abundance increase after 5?min of light. Of these, 84% were bona?fide surface proteins while only 1% of all non-enriched proteins were surface associated (Fig.?2e). We thereby specifically retrieved 43 of the 215 CSPA-reported surfaceome members for this cell line including non-glycosylated surface proteins that typically evade CSC- or LRC-based surfaceome interrogations (Supplementary Fig.?3). LUX-MS, therefore, enables unbiased spatial proteotyping with sub-surfaceome resolution. In a second experiment using replicates, we used statistical testing to identify bona?fide SOG-proximal candidates (enrichment fold change 1.5 and value ?0.05, Fig.?2e and Supplementary Data?1). Among the top hits, we found immunoglobulin G2 (IgG2) isoforms of the Ab-SOG, the primary binding target CD20, human leukocyte antigen class II, tetraspanin CD37, CD298, and CD71 that are actually or functionally associated with CD20 on the surface of B-lymphoma cells12,54C61. Furthermore, LUX-MS revealed the proximity of CD20 to the IgM BCR complex (CD79A, CD79B, IgM heavy, and light chain) and BCR-associated CD19, CD38, and ITGA4 in line with a model where rituximab binding to CD20 is able to eradicate continuously activated B-lymphoma cells by altering survival-promoting BCR signaling51. On resting human B cells that express lower levels of CD20, we found CD20 in proximity to the BCR in presence of CD40 and partial absence of IgM indicating its segregation to quiescent IgD BCR nanoclusters as demonstrated previously51 (Supplementary Fig.?4). Performing LUX-MS in D2O recapitulated these results and, in addition, provided CD20-proximity candidates that were not identified in H2O alone, thereby increasing the number of CD20-proximity candidates from 118 to 172 (Fig.?2d and Supplementary Fig.?4). In contrast, CD20-proximity labeling using HRP-coupled antibodies yielded 220 candidates with a partially overlapping set, including the target receptor and interactors. Furthermore, using SOG-coupled antibodies against CD38, CD54, CD166, and Benzethonium Chloride CD220 revealed distinct receptor-specific surfaceome neighborhoods that covered a wide range of protein functionalities (Fig.?2f). LUX-MS, therefore, enables the discovery of cell-type-specific surfaceome microenvironments of selected receptors on living cells with fine-tunable spatial precision and as.
November 20, 2022