Objective Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but uncovering disease-relevant autoantigens has been a difficult challenge. Our goal was to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, which is thought to result from infection-induced autoimmunity. Methods Using tandem mass spectrometry, naturally presented HLA-DR self-peptides from a patient’s synovium were identified, synthesized and reacted with his peripheral blood mononuclear cells (PBMC). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patients’ cells or sera. Results Of 120 HLA-DR-presented self-peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMC to proliferate. We then found that T and B cell responses to ECGF occurred systemically in about 10–30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. In non-antibiotic-treated historic patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis. Conclusion T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence for autoimmune T and B cell responses in this illness.

HLA-DR-molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1,593 non-redundant HLA-DR-presented peptides, derived from 870 source proteins. Sixty-seven percent of the peptides identified in SFMC and 55% of those found in PBMC were found in ST, but analysis of SFMC/PBMC also revealed new antigen-presented peptides. Peptides were synthesized and examined for reactivity with the patients’ PBMC. To date, three autoantigens in RA and four novel autoantigens in LA, presented in ST and/or PBMC, were shown to be targets of T- and B-cell responses in these diseases; ongoing analyses may add to this list. Thus, immunoprecipitation and LC-MS/MS can now identify hundreds of HLA-DR-presented self-peptides from individual patients’ tissues or fluids with mixed cell populations. Importantly, identification of HLA-DR-presented peptides from SFMC or PBMC allows testing of more patients, including those early in the disease. Direct analysis of clinical samples facilitates identification of novel immunogenic T-cell epitopes. Data are available via ProteomeXchange (identifier PXD003051).

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyze changes in protein expressions in response to ET-1 in pulmonary arterial smooth muscle cells (PASMC) from a healthy Control (non-PAH) and a PAH subject presenting a bone morphogenetic protein type II receptor (BMPR2) mutation with exon 1–8 deletion. Protein expressions were analyzed by proteomic mass spectrometry using label-free quantitation and the correlations were subjected to Ingenuity™ Pathway Analysis. The results point to eIF2/mTOR/p70S6K, RhoA/actin cytoskeleton/integrin and protein unbiquitination as canonical pathways whose protein expressions increase with the development of PAH. These pathways have an intimal function in the PAH-related physiology of smooth muscle proliferation, apoptosis, contraction and cellular stress. Exposure of the cells to ET-1 further increases protein expression within these pathways. Thus our results show changes in signaling pathways as a consequence of PAH and the effect of ET-1 interference on Control and PAH-affected cells.

Sirtuin-1 (SirT1) catalyzes NAD+-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regulate SirT1 activity.Technical barriers against measuring endogenous SirT1 activity have limited characterization of SirT1 in disease and its activation by small molecules. Herein, we developed a relative quantitative mass spectrometry-based technique for measuring endogenous SirT1 activity (RAMSSAY/RelAtive Mass Spectrometry Sirt1 Activity assaY) in cell and tissue homogenates using a biotin-labeled, acetylated p53-derived peptide as a substrate.We demonstrate that oxidative and metabolic stress diminish SirT1 activity in the hepatic cell line HepG2. Moreover, pharmacologic molecules including nicotinamide and EX-527 attenuate SirT1 activity; purported activators of SirT1, the polyphenol S17834, the polyphenol resveratrol, or the non-polyphenolic Sirtris compound SRT1720, failed to activate endogenous SirT1 significantly. Furthermore, we provide evidence that feeding a high fat high sucrose diet (HFHS) to mice inhibits endogenous SirT1 activity in mouse liver.In summary, we introduce a robust, specific and sensitive mass spectrometry-based assay for detecting and quantifying endogenous SirT1 activity using a biotin-labeled peptide in cell and tissue lysates. With this assay, we determine how pharmacologic molecules and metabolic and oxidative stress regulate endogenous SirT1 activity. The assay may also be adapted for other sirtuin isoforms.