Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Structures of six reactivity probes. MSD-DTB, maleimide-sulfonate-dibenzocyclooctyne-DTB. MS-DTB, maleimide-sulfonate-DTB. DBIA, desthiobiotin iodoacetamide. IA-DTB, iodoacetamide-PEG-desthiobiotin. CA-DTB, chloroacetamide-PEG-desthiobiotin. IA-DTB-COOH, iodoacetamide-carboxylate-PEG-desthiobiotin. b MSD-DTB and MS-DTB probes exhibited greater cell surface labeling compared to the others in BV173 and MT2 cells. The result is a representative of three experiments ( n = 3 independent replicates). Fluorescein isothiocyanate (FITC)-A represents the fluorescence intensity of FITC. c . LC-MS analyses revealed that MSD-DTB and MS-DTB probes modify cysteine, but not histidine, serine, and N-terminal amine. The result is a representative of two experiments ( n = 2 independent replicates). d Structures of MD (maleimide-dibenzocyclooctyne), MSD (maleimide-sulfonate-dibenzocyclooctyne), and MSD4 (maleimide-sulfonate-dibenzocyclooctyne-PEG4). e In-gel fluorescence analyses revealed that sulfonated maleimide probes minimally labeled proteins in live cells, while yielding comparable levels of proteome labeling to its cell-permeable counterpart probe, MD, in cell lysates. The result is a representative of two experiments ( n = 2 independent replicates). f Cysteine-directed activity-based protein profiling (ABPP) analyses revealed that MS-DTB modified five cysteines with >20% engagement out of 15,310 quantified cysteines in live cells, while it modified 1730 cysteines with >20% engagement in cell lysates. Data represent mean values ( n = 2 independent replicates). Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Labeling, Fluorescence, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Modification

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: 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.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Flow Cytometry, Knock-Out, Staining, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Schematic representation of the immunopeptidomics workflow. b The number of 8-13-mer MHC-I-associated peptides in MT2 and BV173 cells treated with 50 µM of MS-DTB. The result is a representative of two experiments ( n = 2 independent replicates). c Motif analysis of all 9-mer MHC-I-bound antigens, cysteine-containing 9-mer MHC-I-bound antigens, cysteinylated 9-mer MHC-I-bound antigens, and MS-DTB-modified 9-mer MHC-I-bound antigens. d Distribution of all cysteines, cysteinylated cysteines, and MS-DTB-modified cysteines on 9-mer peptide antigens in MT2 and BV173 cells. e Venn diagram of MHC-I-bound antigens with unmodified, cysteinylated, and MS-DTB-modified cysteines. Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Modification

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Schematic representation of comparative analysis in interferon gamma (IFNγ)-stimulated versus non-stimulated BV173 cells. b Western blot analysis of MHC-I and PSMB9 expression in BV173 cells stimulated with IFNγ (50 ng/mL, 24 h). The result is a representative of two experiments ( n = 2 independent replicates). c ELISA assay measuring the presence of MS-DTB in the pMHC-I complex with or without IFNγ stimulation in BV173 cells. Data represent mean values ± SEM ( n = 3 independent replicates). The statistical significance was evaluated through unpaired two-tailed Student’s t -tests. P value was 0.025. d The number of 8-13-mer MHC-I-associated peptides in IFNγ-stimulated versus non-stimulated cells. e . Motif analysis of all 9-mer MHC-I-bound antigens and MS-DTB-modified MHC-I-bound antigens. f Distribution of MS-DTB-modified cysteines on 9-mer MHC-I-bound antigens in IFNγ-stimulated versus non-stimulated cells. g Heatmap showing the ratio values of amino acid (aa) frequency in IFNγ-stimulated versus non-stimulated cells. The percentage of each aa relative to all amino acids at each position was calculated to derive the ratio values between IFNγ-stimulated and non-stimulated cells. A gray shade indicates aa with a percentage below 2%. Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Modification