Strategies for Top-Down Hydrogen Deuterium Exchange-Mass Spectrometry: A Mini Review and Perspective.

Hydrogen deuterium-exchange mass spectrometry (HDX-MS) is commonly used in the study of protein dynamics and protein interactions. By measuring the isotopic exchange of backbone amide hydrogens in solution, HDX-MS offers valuable structural insights into challenging biological systems. Traditional HDX-MS approaches utilize bottom-up (BU) proteomics, in which deuterated proteins are digested before MS analysis.

Ancestry-based differences in the immune phenotype are associated with lupus activity.

Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities.

The Deuterium Calculator: An Open-Source Tool for Hydrogen-Deuterium Exchange Mass Spectrometry Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time-consuming. Moreover, the HDX community is expanding to investigate multiprotein and highly complex protein systems which further complicates data analysis. Thus, a simple, open-source software package designed to analyze large and highly complex protein systems is needed.

Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients.

Current and Future Biomarkers in Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating autoimmune disorder. Currently, there is a lack of effective treatment for the progressive form of MS, partly due to insensitive readout for neurodegeneration. The recent development of sensitive assays for neurofilament light chain (NfL) has made it a potential new biomarker in predicting MS disease activity and progression, providing an additional readout in clinical trials.

Biomarker panel increases accuracy for identification of an MS relapse beyond sNfL.

Background: For relapsing-remitting multiple sclerosis (RRMS), there is a need for biomarker development beyond clinical manifestations and MRI. Soluble neurofilament light chain (sNfL) has emerged as a biomarker for inflammatory activity in RRMS. However, there are limitations to the accuracy of sNfL in identifying relapses.

Internalization of Polymeric Bacterial Peptidoglycan Occurs through Either Actin or Dynamin Dependent Pathways.

Peptidoglycan (PGN), a polymeric glycan macromolecule, is a major constituent of the bacterial cell wall and a conserved pathogen-associated molecular pattern (PAMP) that triggers immune responses through cytosolic sensors. Immune cells encounter both PGN polymers and hydrolyzed muropeptides during infections, and primary human innate immune cells respond better to polymeric PGN than the minimal bioactive subunit muramyl dipeptide (MDP). While MDP is internalized through macropinocytosis and/or clathrin-mediated endocytosis, the internalization of particulate polymeric PGN is unresolved.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen.

Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.

Interferon-β Intensifies Interleukin-23-Driven Pathogenicity of T Helper Cells in Neuroinflammatory Disease.

Interferon (IFN)-β is a popular therapy for multiple sclerosis (MS). However, 25-40% of patients are nonresponsive to this therapy, and it worsens neuromyelitis optica (NMO), another neuroinflammatory disease. We previously identified, in both NMO patients and in mice, that IFN-β treatment had inflammatory effects in T Helper (TH) 17-induced disease through the production of the inflammatory cytokine IL-6.

Insufficient Anthrax Lethal Toxin Neutralization Is Associated with Antibody Subclass and Domain Specificity in the Plasma of Anthrax-Vaccinated Individuals.

Anthrax vaccine adsorbed (AVA) is a significant line of defense against bioterrorist attack from spores. However, in a subset of individuals, this vaccine may produce a suboptimal quantity of anti-protective antigen (PA), antibodies that are poorly neutralizing, and/or antibody titers that wane over time, necessitating annual boosters. To study individuals with such poor responses, we examine the properties of anti-PA in a subset of vaccinated individuals that make significant quantities of antibody but are still unable to neutralize toxin.

The Role of B Cells in Primary Progressive Multiple Sclerosis.

The success of ocrelizumab in reducing confirmed disability accumulation in primary progressive multiple sclerosis (PPMS) via CD20-targeted depletion implicates B cells as causal agents in the pathogenesis of PPMS. This review explores the possible mechanisms by which B cells contribute to disease progression in PPMS, specifically exploring cytokine production, antigen presentation, and antibody synthesis. B cells may contribute to disease progression in PPMS through cytokine production, specifically GM-CSF and IL-6, which can drive naïve T-cell differentiation into pro-inflammatory Th1/Th17 cells.

Pertussis Toxin Inhibits Encephalitogenic T-Cell Infiltration and Promotes a B-Cell-Driven Disease during Th17-EAE.

Pertussis toxin (PTX) is a required co-adjuvant for experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin antigen. However, PTX's effects on EAE induced by the transfer of myelin-specific T helper cells is not known. Therefore, we investigated how PTX affects the Th17 transfer EAE model (Th17-EAE).

Quantitative Top-Down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling.

Labeling approaches using isobaric chemical tags (e.g., isobaric tagging for relative and absolute quantification, iTRAQ and tandem mass tag, TMT) have been widely applied for the quantification of peptides and proteins in bottom-up MS.

Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry for Metabolite Analysis in Single Cells.

Single-cell capillary electrophoresis mass spectrometry (CE-MS) is a promising platform to analyze cellular contents and probe cell heterogeneity. However, current single-cell CE-MS methods often rely on offline microsampling processes and may demonstrate low sampling precision and accuracy. We have recently developed an electrospray-assisted device, , for low-volume sample extraction.

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis.

Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult.

C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils.

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of , the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown.

Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype.

Background: Autoimmune diseases comprise a spectrum of illnesses and are on the rise worldwide. Although antinuclear antibodies (ANAs) are detected in many autoimmune diseases, up to 20% of healthy women are ANA-positive (ANA+) and most will never develop clinical symptoms. Furthermore, disease transition is higher among ANA+ African Americans compared with ANA+ European Americans.

Spray-Capillary: An Electrospray-Assisted Device for Quantitative Ultralow-Volume Sample Handling.

The analysis of low-volume samples provides valuable insight into complex biological systems. However, the proteomic and metabolomic analysis of low-volume samples remains challenging due to the lack of simple, efficient, and reproducible microsampling techniques. We have developed an electrospray-assisted device for quantitative low-volume sample extraction, referred to here as "Spray-Capillary".

B cell function impacts the efficacy of IFN-β therapy in EAE.

Recent studies identified that interferon beta (IFN-β) treatment skews B-cells towards a regulatory phenotype in multiple sclerosis. To assess B cell involvement during IFN-β therapy, we compared IFN-β treatment in a B cell-independent model and a B cell-dependent model of experimental autoimmune encephalomyelitis (EAE). We show that in B cell-independent EAE, IFN-β ameliorates neuroinflammation.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population.

Monocyte procoagulant responses to anthrax peptidoglycan are reinforced by proinflammatory cytokine signaling.

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis.