Comparison of the MHC I Immunopeptidome Repertoire of B-Cell Lymphoblasts Using Two Isolation Methods.

Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, a comparison of these approaches for the isolation of two different B-cell lymphoblast cell models is presented, and it is reported on the recovery, reproducibility, scalability, and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4-fold increase in MIPs compared to the MAE. The comprehensiveness of these immunopeptidome analyses is extended using personalized genomic database of B-cell lymphoblasts, and it is discovered that 0.4% of their respective MIP repertoire harbored nonsynonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs).

CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization.

Protein cross-linking mass spectrometry (CL-MS) enables the sensitive detection of protein interactions and the inference of protein complex topology. The detection of chemical cross-links between protein residues can identify intra- and interprotein contact sites or provide physical constraints for molecular modeling of protein structure. Recent innovations in cross-linker design, sample preparation, mass spectrometry, and software tools have significantly improved CL-MS approaches. Although a number of algorithms now exist for the identification of cross-linked peptides from mass spectral data, a dearth of user-friendly analysis tools represent a practical bottleneck to the broad adoption of the approach. To facilitate the analysis of CL-MS data, we developed CLMSVault, a software suite designed to leverage existing CL-MS algorithms and provide intuitive and flexible tools for cross-platform data interpretation. CLMSVault stores and combines complementary information obtained from different cross-linkers and search algorithms. CLMSVault provides filtering, comparison, and visualization tools to support CL-MS analyses and includes a workflow for label-free quantification of cross-linked peptides. An embedded 3D viewer enables the visualization of quantitative data and the mapping of cross-linked sites onto PDB structural models. We demonstrate the application of CLMSVault for the analysis of a noncovalent Cdc34-ubiquitin protein complex cross-linked under different conditions. CLMSVault is open-source software (available at https://gitlab.com/courcelm/clmsvault.git ), and a live demo is available at http://democlmsvault.tyerslab.com/ .

Estradiol inhibits vascular endothelial cells pro-inflammatory activation induced by C-reactive protein.

In addition of being an important inflammatory biomarker and a risk factor for cardiovascular disease, much evidence indicates that the C-reactive protein (CRP) contributes to the atherosclerosis development process. This plasmatic protein synthesized by hepatocytes in response to inflammation and tissue injury induces pro-inflammatory molecules' expression by endothelial cells (ECs). Previous studies showed that the 17ß-estradiol (E2) has beneficial effects on vascular cells by reducing in vitro pro-inflammatory molecules expressions in EC. Therefore, we hypothesize that E2 blocks or reduces CRP-mediated inflammatory responses by modulating endogenous production of CRP in EC and/or activation mechanisms. Using human aortic ECs (HAECs), we first evaluated CRP production by vascular EC and second demonstrated its self-induction. Indeed, recombinant human CRP stimulation induces a fivefold increase of CRP expression. A 1-h pre-treatment of E2 at a physiologic dose (10(-9 )M) leads to an important decrease of CRP production suggesting a partial blockage of its amplification loop mechanism. Furthermore, in HAEC, E2 reduces the secretion of the most potent agonist of CRP induction, the IL-6, by 21 %. E2 pre-treatment also decreased the expression of pro-inflammatory molecules IL-8, VCAM-1, and ICAM-1 induced by CRP and involved in leukocytes recruitment. In addition, we demonstrated that E2 could restore vascular endothelial growth factor-mediated EC migration response impaired by CRP suggesting another pro-angiogenic property of this hormone. These findings suggest that E2 can interfere with CRP pro-inflammatory effects via activation signals using its rapid, non-genomic pathway that may provide a new mechanism to improve vascular repair.