Fig. 2: Sulfonated maleimide probes modify cysteines on MHC-I-bound antigens.
From: A platform for mapping reactive cysteines within the immunopeptidome
a Quantitative global proteomics and flow cytometry studies confirmed the knockout (KO) of HLA class I genes in MT2 cells. Global proteomics data represent mean values (n  =  3 independent replicates). The result of flow cytometry analysis is a representative of two experiments (n = 2 independent replicates). b Flow cytometry analyses measuring Streptavidin-FITC staining on HLA wildtype (WT) and KO cells treated with the MSD-DTB or MS-DTB probe. Data represent mean values (n  =  2 independent replicates in MT2 and MDA-MB-231 cells treated with MSD-DTB, and MDA-MB-231 cells treated with MS-DTB) and mean values ± SEM (n  =  3 independent replicates in BV173 cells treated with MSD-DTB and MS-DTB, and n  =  4 independent replicates in MT2 cells treated with MS-DTB). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. P values were 0.037, 0.0029, and 0.048. c Quantitative global proteomics and flow cytometry studies confirmed the complete knockout of HLA class I genes in MT2 cells. Global proteomics data represent mean values (n  =  3 independent replicates). P values were calculated by two-sided t test and adjusted using Benjamini-Hochberg correction for multiple comparisons. The result of flow cytometry analysis is a representative of two experiments (n = 2 independent replicates). d Flow cytometry analyses measuring Streptavidin-FITC staining on MT2 parental and HLA knockout cells treated with MS-DTB. Data represent mean values ± SEM (n  =  4 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. P values were 0.0029 and 9.2 × 10−6. e Structure of NHS-sulfonate-biotin and flow cytometry analyses measuring Streptavidin-FITC staining on BV173 HLA wildtype and knockout cells treated with NHS-sulfonate-biotin. Data represent mean values (n  =  2 independent replicates). NHS, N-hydroxysuccinimide. f ELISA assay measuring MS-DTB in the pMHC-I complex. Data represent mean values ± SEM (n  =  3 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. P values were 8.1 × 10−5, 0.00028, 6.2 × 10−6, and 8.7 × 10−6. HRP, horseradish peroxidase. g ELISA assay measuring MHC-I and β2-microglobulin (β2M) assembly. Data represent mean values ± SEM (n  =  3 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. Source data are provided as a Source Data file.
