Journal: Nature Communications

Article Title: Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

doi: 10.1038/s41467-021-27280-x

Figure Lengend Snippet: a Schematic of decorating an antibody (Ab) with the singlet oxygen generator (SOG) thiorhodamine. b Flow cytometric analysis of human peripheral blood mononuclear cells (PBMC) stained for cell surface biotin and immunoglobulin before and after LUX-labeling with anti-CD20 antibody-SOG conjugate (anti-CD20-SOG) (>60,000 cells per condition). c Histogram plot showing light-dependent cell surface biotinylation of LUX-labeled B-lymphoma SUDHL6 cells (>50,000 cells per condition). d Scatter plot showing light-dependent enrichment of LUX-MS quantified proteins from anti-CD20-SOG treated B-lymphoma SUDHL6 cells. e Fraction of surface proteins of LUX-MS quantified proteins found to be 5-fold enriched or not enriched after illumination of anti-CD20-SOG-treated B-lymphoma SUDHL6 cells. f Left, Volcano plot showing relative abundance changes of LUX-MS quantified proteins from anti-CD20-SOG treated B-lymphoma SUDHL6 cells with and without illumination for 5 min, tested using a two-sided Student’s t test. Green and blue dots represent known and previously unknown CD20 associated proteins, respectively. Orange and red dots represent chains of the used antibody and the primary binding target CD20. Right, literature and LUX-MS-based cell surface interaction network of CD20. g Venn diagram showing overlap of CD20 proximal candidates identified on resting human B cells (Ramos) using horseradish peroxidase (HRP) conjugated antibodies or LUX-MS performed once in water (H 2 O) or heavy water (D 2 O) 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 ).

Article Snippet: Patient-derived B-lymphoma cell line SUDHL6 (ATCC, CRL-2959) and human Burkitt lymphoma B cell-line Ramos (ATCC, CRL-1596, kindly provided by Michael Reth) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 1.5 mM GlutaMAX, 1% penicillin–streptomycin, and 10% fetal bovine serum.

Techniques: Staining, Labeling, Binding Assay

Journal: Nature Communications

Article Title: Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

doi: 10.1038/s41467-021-27280-x

Figure Lengend Snippet: a Schematic of coupling the small-molecule drug cardiac glycoside CG1 to the singlet oxygen generator (SOG) thiorhodamine. b Single-cell chemosensitivity screen showing the viability of human promyelocytic leukemia (HL60) cells after incubation with free CG1, CG1 coupled to thiorhodamine (CG1-SOG) or horseradish peroxidase (CG1-HRP) for 48 h. Data are presented as mean values ±SD ( n = 10 technical replicates). c Volcano plot showing relative abundance changes of LUX-MS quantified proteins from CG1-SOG treated promyelocytic leukemia HL60 cells with and without illumination for 5 min, tested using a two-sided Student’s t test. Dots and crosses represent cell surface and otherwise annotated proteins, respectively. Red, green and blue dots represent the binding target of CG1, known and previously unknown surfaceome interactors, respectively. The former two are highlighted. d Schematic of coupling the biomolecules insulin and transferrin to the singlet oxygen generator (SOG) thiorhodamine. e Volcano plot showing relative abundance changes of LUX-MS quantified proteins from insulin-SOG and transferrin-SOG treated B-lymphoma SUDHL6 cells illuminated for 5 min, tested using a two-sided Student’s t test. Dots and crosses represent cell surface and otherwise annotated proteins, respectively. Brown, red and blue dots represent the ligand, primary binding target, and potential surfaceome interactors, respectively. The former two are highlighted together with known surfaceome interactors. f Schematic representation of the surfaceome proximity network identified by LUX-MS. Source data are provided as a Source Data file and interactive volcano plots (Supplementary Data and ).

Article Snippet: Patient-derived B-lymphoma cell line SUDHL6 (ATCC, CRL-2959) and human Burkitt lymphoma B cell-line Ramos (ATCC, CRL-1596, kindly provided by Michael Reth) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 1.5 mM GlutaMAX, 1% penicillin–streptomycin, and 10% fetal bovine serum.

Techniques: Incubation, Binding Assay