Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains

Crosstalk between Medulloblastoma Cells and Endothelium Triggers a Strong Chemotactic Signal Recruiting T Lymphocytes to the Tumor Microenvironment

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)” is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant “Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES).” This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications

Regards de physiciens sur des systemes moleculaires d'interet biologique

IP

IRMPD spectroscopy and QC calculations on functionalized gold nanocluster ions

International audienc

Design, optimisation and standardisation of a high‐dimensional spectral flow cytometry workflow assessing T‐cell immunophenotype in patients with melanoma

Abstract Objectives Despite the success of immune checkpoint blockade, most metastatic melanoma patients fail to respond to therapy or experience severe toxicity. Assessment of biomarkers and immunophenotypes before or early into treatment will help to understand favourable responses and improve therapeutic outcomes. Methods We present a high‐dimensional approach for blood T‐cell profiling using three multi‐parameter cytometry panels: (1) a TruCount panel for absolute cell counts, (2) a 27‐colour spectral panel assessing T‐cell markers and (3) a 20‐colour spectral panel evaluating intracellular cytokine expression. Pre‐treatment blood mononuclear cells from patients and healthy controls were cryopreserved before staining across 11 batches. Batch effects were tracked using a single‐donor control and the suitability of normalisation was assessed. The data were analysed using manual gating and high‐dimensional strategies. Results Batch‐to‐batch variation was minimal, as demonstrated by the dimensionality reduction of batch‐control samples, and normalisation did not improve manual or high‐dimensional analysis. Application of the workflow demonstrated the capacity of the panels and showed that patients had fewer lymphocytes than controls (P = 0.0027), due to lower naive CD4+ (P = 0.015) and CD8+ (P = 0.011) T cells and follicular helper T cells (P = 0.00076). Patients showed trends for higher proportions of Ki67 and IL‐2‐expressing cells within CD4+ and CD8+ memory subsets, and increased CD57 and EOMES expression within TCRγδ+ T cells. Conclusion Our optimised high‐parameter spectral cytometry approach provided in‐depth profiling of blood T cells and found differences in patient immunophenotype at baseline. The robustness of our workflow, as demonstrated by minimal batch effects, makes this approach highly suitable for the longitudinal evaluation of immunotherapy effects