The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

The coevolution of liquid chromatography (LC) with mass spectrometry (MS) has shaped contemporary proteomics. LC hyphenated to MS now enables quantification of more than 10,000 proteins in a single injection, a number that likely represents most proteins in specific human cells or tissues. Separations by ion mobility spectrometry (IMS) have recently emerged to complement LC and further improve the depth of proteomics. Given the theoretical advantages in speed and robustness of IMS in comparison to LC, we envision that ongoing improvements to IMS paired with MS may eventually make LC obsolete, especially when combined with targeted or simplified analyses, such as rapid clinical proteomics analysis of defined biomarker panels. In this perspective, we describe the need for faster analysis that might drive this transition, the current state of direct infusion proteomics, and discuss some technical challenges that must be overcome to fully complete the transition to entirely gas phase proteomics.

Insights into the study and origin of the citrullinome in rheumatoid arthritis.

The presence of autoantibodies and autoreactive T cells to citrullinated proteins and citrullinating enzymes in patients with rheumatoid arthritis (RA), together with the accumulation of citrullinated proteins in rheumatoid joints, provides substantial evidence that dysregulated citrullination is a hallmark feature of RA. However, understanding mechanisms that dysregulate citrullination in RA has important challenges. Citrullination is a normal process in immune and non-immune cells, which is likely activated by different conditions (eg, inflammation) with no pathogenic consequences. In a complex inflammatory environment such as the RA joint, unique strategies are therefore required to dissect specific mechanisms involved in the abnormal production of citrullinated proteins. Here, we will review current models of citrullination in RA and discuss critical components that, in our view, are relevant to understanding the accumulation of citrullinated proteins in the RA joint, collectively referred to as the RA citrullinome. In particular, we will focus on potential caveats in the study of citrullination in RA and will highlight methods to precisely detect citrullinated proteins in complex biological samples, which is a confirmatory approach to mechanistically link the RA citrullinome with unique pathogenic pathways in RA.

BIRCH: An Automated Workflow for Evaluation, Correction, and Visualization of Batch Effect in Bottom-Up Mass Spectrometry-Based Proteomics Data.

Recent surges in large-scale mass spectrometry (MS)-based proteomics studies demand a concurrent rise in methods to facilitate reliable and reproducible data analysis. Quantification of proteins in MS analysis can be affected by variations in technical factors such as sample preparation and data acquisition conditions leading to batch effects, which adds to noise in the data set. This may in turn affect the effectiveness of any biological conclusions derived from the data. Here we present Batch-effect Identification, Representation, and Correction of Heterogeneous data (BIRCH), a workflow for analysis and correction of batch effect through an automated, versatile, and easy to use web-based tool with the goal of eliminating technical variation. BIRCH also supports diagnosis of the data to check for the presence of batch effects, feasibility of batch correction, and imputation to deal with missing values in the data set. To illustrate the relevance of the tool, we explore two case studies, including an iPSC-derived cell study and a Covid vaccine study to show different context-specific use cases. Ultimately this tool can be used as an extremely powerful approach for eliminating technical bias while retaining biological bias, toward understanding disease mechanisms and potential therapeutics.

Serological response to vaccination in post-acute sequelae of COVID.

BACKGROUND: Individuals with post-acute sequelae of COVID (PASC) may have a persistence in immune activation that differentiates them from individuals who have recovered from COVID without clinical sequelae. To investigate how humoral immune activation may vary in this regard, we compared patterns of vaccine-provoked serological response in patients with PASC compared to individuals recovered from prior COVID without PASC. METHODS: We prospectively studied 245 adults clinically diagnosed with PASC and 86 adults successfully recovered from prior COVID. All participants had measures of humoral immunity to SARS-CoV-2 assayed before or after receiving their first-ever administration of COVID vaccination (either single-dose or two-dose regimen), including anti-spike (IgG-S and IgM-S) and anti-nucleocapsid (IgG-N) antibodies as well as IgG-S angiotensin-converting enzyme 2 (ACE2) binding levels. We used unadjusted and multivariable-adjusted regression analyses to examine the association of PASC compared to COVID-recovered status with post-vaccination measures of humoral immunity. RESULTS: Individuals with PASC mounted consistently higher post-vaccination IgG-S antibody levels when compared to COVID-recovered (median log IgG-S 3.98 versus 3.74, P < 0.001), with similar results seen for ACE2 binding levels (median 99.1 versus 98.2, P = 0.044). The post-vaccination IgM-S response in PASC was attenuated but persistently unchanged over time (P = 0.33), compared to in COVID recovery wherein the IgM-S response expectedly decreased over time (P = 0.002). Findings remained consistent when accounting for demographic and clinical variables including indices of index infection severity and comorbidity burden. CONCLUSION: We found evidence of aberrant immune response distinguishing PASC from recovered COVID. This aberrancy is marked by excess IgG-S activation and ACE2 binding along with findings consistent with a delayed or dysfunctional immunoglobulin class switching, all of which is unmasked by vaccine provocation. These results suggest that measures of aberrant immune response may offer promise as tools for diagnosing and distinguishing PASC from non-PASC phenotypes, in addition to serving as potential targets for intervention.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

An Update on Diagnostic Laboratory Biomarkers for Multiple Sclerosis.

PURPOSE: For many patients, the multiple sclerosis (MS) diagnostic process can be lengthy, costly, and fraught with error. Recent research aims to address the unmet need for an accurate and simple diagnostic process through discovery of novel diagnostic biomarkers. This review summarizes recent studies on MS diagnostic fluid biomarkers, with a focus on blood biomarkers, and includes discussion of technical limitations and practical applicability. RECENT FINDINGS: This line of research is in its early days. Accurate and easily obtainable biomarkers for MS have not yet been identified and validated, but several approaches to uncover them are underway. Continue efforts to define laboratory diagnostic biomarkers are likely to play an increasingly important role in defining MS at the earliest stages, leading to better long-term clinical outcomes.

Discovery Proteomics for COVID-19: Where We Are Now.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the pandemic coronavirus disease 2019 (COVID-19), which has had a devastating impact on society. Here, we summarize proteomic research that has helped elucidate hallmark proteins associated with the disease with respect to both short- and long-term diagnosis and prognosis. Additionally, we review the highly variable humoral response associated with COVID-19 and the increased risk of autoimmunity.

Paradoxical sex-specific patterns of autoantibody response to SARS-CoV-2 infection.

BACKGROUND: Pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating the acute response and recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, the aim of the study was to examine sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. METHODS: In this study, we assessed autoantibody (AAB) reactivity to 91 autoantigens previously linked to a range of classic autoimmune diseases in a cohort of 177 participants (65% women, 35% men, mean age of 35) with confirmed evidence of prior SARS-CoV-2 infection based on presence of antibody to the nucleocapsid protein of SARS-CoV-2. Data were compared to 53 pre-pandemic healthy controls (49% women, 51% men). For each participant, socio-demographic data, serological analyses, SARS-CoV-2 infection status and COVID-19 related symptoms were collected by  an electronic survey of questions. The symptoms burden score was constructed based on the total number of reported symptoms (N = 21) experienced within 6 months prior to the blood draw, wherein a greater number of symptoms corresponded to a higher score and assigned as more severe burden. RESULTS: In multivariable analyses, we observed sex-specific patterns of autoreactivity associated with the presence or absence (as well as timing and clustering of symptoms) associated with prior COVID-19 illness. Whereas the overall AAB response was more prominent in women following asymptomatic infection, the breadth and extent of AAB reactivity was more prominent in men following at least mildly symptomatic infection. Notably, the observed reactivity included distinct antigens with molecular homology with SARS-CoV-2. CONCLUSION: Our results reveal that prior SARS-CoV-2 infection, even in the absence of severe clinical disease, can lead to a broad AAB response that exhibits sex-specific patterns of prevalence and antigen selectivity. Further understanding of the nature of triggered AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19.

Role of protein deimination in cardiovascular diseases: potential new avenues for diagnostic and prognostic biomarkers.

INTRODUCTION: Arginine deimination (citrullination) is a post-translational modification catalyzed by a family of peptidyl arginine deiminase (PAD) enzymes. Cell-based functional studies and animal models have manifested the key role of PADs in various cardiovascular diseases (CVDs). AREA COVERED: This review summarizes the past 10 years of knowledge on the role of PADs in CVD pathogenesis. It focuses on the PAD functions and diverse citrullinated proteins in cardiovascular conditions like deep vein thrombosis, ischemia/reperfusion, and atherosclerosis. Identification of PAD isoforms and citrullinated targets are essential for directing diagnosis and clinical intervention. Finally, anti-citrullinated protein antibodies (ACPAs) are addressed as an independent risk factor for cardiovascular events. EXPERT OPINION: PAD is an unique family of enzymes that permanently converts amino acid arginine to amino acid citrulline in protein . Overexpression or increased activity of PAD has been observed in various CVDs with acute and chronic inflammation as the background. Importantly, far beyond being simply involved in forming neutrophil extracellular traps (NETs), accumulating evidence indicated PAD activation as a trigger for numerous processes, such as transcriptional regulation, endothelial dysfunction, and thrombus formation. In summary, the findings so far have testified the important role of deimination in cardiovascular biology, while more basic and translational studies are essential for further exploration.

Symptomology following mRNA vaccination against SARS-CoV-2.

Despite demonstrated efficacy of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease-2019 (COVID-19), widespread hesitancy to vaccination persists. Improved knowledge regarding frequency, severity, and duration of vaccine-associated symptoms may help reduce hesitancy. In this prospective observational study, we studied 1032 healthcare workers who received both doses of the Pfizer-BioNTech SARS-CoV-2 mRNA vaccine and completed post-vaccine symptom surveys both after dose 1 and after dose 2. We defined appreciable post-vaccine symptoms as those of at least moderate severity and lasting at least 2 days. We found that symptoms were more frequent following the second vaccine dose than the first (74% vs. 60%, P < 0.001), with >80% of all symptoms resolving within 2 days. The most common symptom was injection site pain, followed by fatigue and malaise. Overall, 20% of participants experienced appreciable symptoms after dose 1 and 30% after dose 2. In multivariable analyses, female sex was associated with greater odds of appreciable symptoms after both dose 1 (OR, 95% CI 1.73, 1.19-2.51) and dose 2 (1.76, 1.28-2.42). Prior COVID-19 was also associated with appreciable symptoms following dose 1, while younger age and history of hypertension were associated with appreciable symptoms after dose 2. We conclude that most post-vaccine symptoms are reportedly mild and last <2 days. Appreciable post-vaccine symptoms are associated with female sex, prior COVID-19, younger age, and hypertension. This information can aid clinicians in advising patients on the safety and expected symptomatology associated with vaccination.

Precision Profiling of the Cardiovascular Post-Translationally Modified Proteome: Where There Is a Will, There Is a Way.

There is an exponential increase in biological complexity as initial gene transcripts are spliced, translated into amino acid sequence, and post-translationally modified. Each protein can exist as multiple chemical or sequence-specific proteoforms, and each has the potential to be a critical mediator of a physiological or pathophysiological signaling cascade. Here, we provide an overview of how different proteoforms come about in biological systems and how they are most commonly measured using mass spectrometry-based proteomics and bioinformatics. Our goal is to present this information at a level accessible to every scientist interested in mass spectrometry and its application to proteome profiling. We will specifically discuss recent data linking various protein post-translational modifications to cardiovascular disease and conclude with a discussion for enablement and democratization of proteomics across the cardiovascular and scientific community. The aim is to inform and inspire the readership to explore a larger breadth of proteoform, particularity post-translational modifications, related to their particular areas of expertise in cardiovascular physiology.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure.

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.

Mining the Proteome Associated with Rheumatic and Autoimmune Diseases.

A steady increase in the incidence of osteoarthritis and other rheumatic diseases has been observed in recent decades, including autoimmune conditions such as rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, systemic sclerosis, and Sjögren's syndrome. Rheumatic and autoimmune diseases (RADs) are characterized by the inflammation of joints, muscles, or other connective tissues. In addition to often experiencing debilitating mobility and pain, RAD patients are also at a higher risk of suffering comorbidities such as cardiovascular or infectious events. Given the socioeconomic impact of RADs, broad research efforts have been dedicated to these diseases worldwide. In the present work, we applied literature mining platforms to identify "popular" proteins closely related to RADs. The platform is based on publicly available literature. The results not only will enable the systematic prioritization of candidates to perform targeted proteomics studies but also may lead to a greater insight into the key pathogenic processes of these disorders.

Mapping Citrullinated Sites in Multiple Organs of Mice Using Hypercitrullinated Library.

Protein citrullination (or deimination), an irreversible post-translational modification, has been implicated in several physiological and pathological processes, including gene expression regulation, apoptosis, rheumatoid arthritis, and Alzheimer's disease. Several research studies have been carried out on citrullination under many conditions. However, until now, challenges in sample preparation and data analysis have made it difficult to confidently identify a citrullinated protein and assign the citrullinated site. To overcome these limitations, we generated a mouse hyper-citrullinated spectral library and set up coordinates to confidently identify and validate citrullinated sites. Using this workflow, we detect a four-fold increase in citrullinated proteome coverage across six mouse organs compared with the current state-of-the art techniques. Our data reveal that the subcellular distribution of citrullinated proteins is tissue-type-dependent and that citrullinated targets are involved in fundamental physiological processes, including the metabolic process. These data represent the first report of a hyper-citrullinated library for the mouse and serve as a central resource for exploring the role of citrullination in this organism.

Association of anti-citrullinated protein or peptide antibodies with left ventricular structure and function in rheumatoid arthritis.

Objective: High levels of ACPAs in RA are associated with more severe arthritis and worse prognosis. However, the role of ACPAs in mediating the increased risk of heart failure in RA remains undefined. We examined whether specific ACPAs were associated with subclinical left ventricular (LV) phenotypes that presage heart failure. Methods: Sera from RA patients without clinical cardiovascular disease were assayed for specific ACPAs using a custom Bio-Plex bead assay, and their cross-sectional associations with cardiac magnetic resonance-derived LV measures were evaluated. High ACPA level was defined as ⩾ 75th percentile. Findings were assessed in a second independent RA cohort with an expanded panel of ACPAs and LV measures assessed by 3D-echocardiography. Results: In cohort 1 (n = 76), higher levels of anti-citrullinated fibrinogen 41-60 and anti-citrullinated vimentin antibodies were associated with a 10 and 6% higher adjusted mean LV mass index (LVMI), respectively, compared with lower antibody levels (P < 0.05). In contrast, higher levels of anti-citrullinated biglycan 247-266 were associated with a 13% lower adjusted mean LVMI compared with lower levels (P < 0.001). In cohort 2 (n = 74), the association between ACPAs targeting citrullinated fibrinogen and citrullinated vimentin peptides or protein and LVMI was confirmed: higher anti-citrullinated fibrinogen 556-575 and anti-citrullinated vimentin 58-77 antibody levels were associated with a higher adjusted mean LVMI (19 and 15%, respectively; P < 0.05), but no association with biglycan was found. Conclusion: Higher levels of antibodies targeting citrullinated fibrinogen and vimentin peptides or protein were associated with a higher mean LVMI in both RA cohorts, potentially implicating autoimmune targeting of citrullinated proteins in myocardial remodelling in RA.

DCM-Spheroid Morphs Express PADs and Citrullinated Cytoskeletal Proteins.

During investigating the role of peptidylarginine deiminase (PAD) enzymes in dilated cardiomyopathy (DCM), we observed unique spheroid formation in DCM-myofibroblasts that distinguished them from normal cardiac myofibroblasts. The present study aimed to assess the presence of PADs, the extracellular matrix (ECM), and citrullination in DCM spheroids using immunofluorescence staining and imaging techniques. The results revealed that spheroids derived from DCM-myofibroblasts displayed a more distinctive, tightly packed structure compared with those derived from human cardiac fibroblasts. DCM spheroids showed abundant protein expression of the PAD 2, 3, and 4 enzymes. Notably, increased Ki67 protein expression was associated with increased proliferation in DCM spheroids. Cytoskeletal proteins such as Col-1A, vimentin, α-SMA, and F-actin were highly abundant in DCM spheroids. Furthermore, DCM spheroids contained citrullinated cytoskeletal proteins, mainly citrullinated vimentin and citrullinated fibronectin. These observations supported the occurrence of PAD-mediated citrullination of ECM proteins in DCM spheroids. Collectively, these findings describe the distinctive features of DCM spheroids, representing the cellular characteristics of DCM myofibroblasts. Therefore, DCM spheroids can serve as an in vitro model for further investigations of disease morphology and therapeutic efficacy.

Identification of retinal tau oligomers, citrullinated tau, and other tau isoforms in early and advanced AD and relations to disease status.

Importance: This study identifies and quantifies diverse pathological tau isoforms in the retina of both early and advanced-stage Alzheimer's disease (AD) and determines their relationship with disease status. Objective: A case-control study was conducted to investigate the accumulation of retinal neurofibrillary tangles (NFTs), paired helical filament (PHF)-tau, oligomeric tau (oligo-tau), hyperphosphorylated tau (p-tau), and citrullinated tau (Cit-tau) in relation to the respective brain pathology and cognitive dysfunction in mild cognitively impaired (MCI) and AD dementia patients versus normal cognition (NC) controls. Design setting and participants: Eyes and brains from donors diagnosed with AD, MCI (due to AD), and NC were collected (n=75 in total), along with clinical and neuropathological data. Brain and retinal cross-sections-in predefined superior-temporal and inferior-temporal (ST/IT) subregions-were subjected to histopathology analysis or Nanostring GeoMx digital spatial profiling. Main outcomes and measure: Retinal burden of NFTs (pretangles and mature tangles), PHF-tau, p-tau, oligo-tau, and Cit-tau was assessed in MCI and AD versus NC retinas. Pairwise correlations revealed associations between retinal and brain parameters and cognitive status. Results: Increased retinal NFTs (1.8-fold, p=0.0494), PHF-tau (2.3-fold, p<0.0001), oligo-tau (9.1-fold, p<0.0001), CitR 209 -tau (4.3-fold, p<0.0001), pSer202/Thr205-tau (AT8; 4.1-fold, p<0.0001), and pSer396-tau (2.8-fold, p=0.0015) were detected in AD patients. Retinas from MCI patients showed significant increases in NFTs (2.0-fold, p=0.0444), CitR 209 -tau (3.5-fold, p=0.0201), pSer396-tau (2.6-fold, p=0.0409), and, moreover, oligo-tau (5.8-fold, p=0.0045). Nanostring GeoMx quantification demonstrated upregulated retinal p-tau levels in MCI patients at phosphorylation sites of Ser214 (2.3-fold, p=0.0060), Ser396 (1.8-fold, p=0.0052), Ser404 (2.4-fold, p=0.0018), and Thr231 (3.3-fold, p=0.0028). Strong correlations were found between retinal tau forms to paired-brain pathology and cognitive status: a) retinal oligo-tau vs. Braak stage (r=0.60, P=0.0002), b) retinal PHF-tau vs. ABC average score (r=0.64, P=0.0043), c) retinal pSer396-tau vs. brain NFTs (r=0.68, P<0.0001), and d) retinal pSer202/Thr205-tau vs. MMSE scores (r= -0.77, P=0.0089). Conclusions and Relevance: This study reveals increases in immature and mature retinal tau isoforms in MCI and AD patients, highlighting their relationship with brain pathology and cognition. The data provide strong incentive to further explore retinal tauopathy markers that may be useful for early detection and monitoring of AD staging through noninvasive retinal imaging.

Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway.

BACKGROUND: Cardiac fibrosis contributes to end-stage extracellular matrix remodeling and heart failure (HF). Cardiac fibroblasts (CFs) differentiate into myofibroblasts (myoFbs) to preserve the structural integrity of the heart; however, the molecular mechanisms regulating CF transdifferentiation remain poorly understood. Protein arginine deiminase (PAD), which converts arginine to citrulline, has been shown to play a role in myocardial infarction, fibrosis, and HF. This study aimed to investigate the role of PAD in CF differentiation to myoFbs and identify the citrullinated proteins that were associated with phenotypic changes in CFs. RESULTS: Gene expression analysis showed that PAD1 and PAD2 isoforms, but not PAD4 isoforms, were abundant in both CFs and myoFbs, and PAD1 was significantly upregulated in myoFbs. The pan-PAD inhibitor BB-Cl-amidine (BB-Cl) downregulated the mRNA expression of PAD1 and PAD2 as well as the protein expression of the fibrosis marker COL1A1 in CFs and myoFbs. Interestingly, a proteomic approach pointed to the activation of the Nrf2/HO-1 signaling pathway upon BB-Cl treatment in CFs and myoFbs. BB-Cl administration resulted in the upregulation of HO-1 at both the gene and protein levels in CFs and myoFbs. Importantly, the protein citrullination landscape of CFs consisting of 86 novel citrullination sites associated with focal adhesion (FN1(R1054)), inflammation (TAGLN(R12)) and DNA replication (EEF2(R767)) pathways was identified. CONCLUSIONS: In summary, we revealed that BB-Cl treatment resulted in increased HO-1 expression via the Nrf2 pathway, which could prevent excessive tissue damage, thereby leading to substantial clinical benefits for the treatment of cardiac fibrosis.

Peptidyl arginine deiminase inhibition alleviates angiotensin II-induced fibrosis.

OBJECTIVES: The conversion of protein arginine residues to citrulline by calcium-dependent peptidyl arginine deiminases (PADs) has been implicated in the pathogenesis of several diseases, indicating that PADs are therapeutic targets. A recent study indicated that PAD4 regulates age-related organ fibrosis and dysfunction; however, the specific role of this PAD and its citrullination substrate remains unclear. We investigated whether pharmacological inhibition of PAD activity could affect the progression of fibrosis and restore heart function. METHODS: Cardiac hypertrophy was induced by chronic infusion of angiotensin (Ang) II. After 2 weeks of AngII infusion, a PAD inhibitor (Cl-amidine hydrochloride) or vehicle (saline) was injected every other day for the next 14 days together with the continued administration of AngII for a total of up to 28 days. Cardiac fibrosis and remodeling were evaluated by quantitative heart tissue histology, echocardiography, and mass spectrometry. RESULTS: A reverse AngII-induced effect was observed in PAD inhibitor-treated mice (n=6) compared with AngII vehicle-treated mice, as indicated by a significant reduction in the heart/body ratio (AngII: 6.51±0.8 mg/g vs. Cl-amidine: 5.27±0.6 mg/g), a reduction in fibrosis (AngII: 2.1-fold increased vs. Cl-amidine: 1.8-fold increased), and a reduction in left ventricular posterior wall diastole (LWVPd) (AngII: 1.1±0.04 vs. Cl-amidine: 0.78±0.02 mm). Label-free quantitative proteomics analysis of heart tissue indicated that proteins involved in fibrosis (e.g., periostin), cytoskeleton organization (e.g., transgelin), and remodeling (e.g., myosin light chain, carbonic anhydrase) were normalized by Cl-amidine treatment. CONCLUSION: Our findings demonstrate that pharmacological inhibition of PAD may be an effective strategy to attenuate cardiac fibrosis.

Protein arginine deiminase 2 (PAD2) modulates the polarization of THP-1 macrophages to the anti-inflammatory M2 phenotype.

BACKGROUND: Macrophages are effector cells of the innate immune system that undergo phenotypical changes in response to organ injury and repair. These cells are most often classified as proinflammatory M1 and anti-inflammatory M2 macrophages. Protein arginine deiminase (PAD), which catalyses the irreversible conversion of protein-bound arginine into citrulline, is expressed in macrophages. However, the substrates of PAD and its role in immune cells remain unclear. This study aimed to investigate the role of PAD in THP-1 macrophage polarization to the M1 and M2 phenotypes and identify the citrullinated proteins and modified arginines that are associated with this biological switch using mass spectrometry. RESULTS: Our study showed that PAD2 and, to a lesser extent, PAD1 and PAD4 were predominantly expressed in M1 macrophages. We showed that inhibiting PAD expression with BB-Cl-amidine decreased macrophage polarization to the M1 phenotype (TNF-α, IL-6) and increased macrophage polarization to the M2 phenotype (MRC1, ALOX15). This process was mediated by the downregulation of proteins involved in the NF-κß pathway. Silencing PAD2 confirmed the activation of M2 macrophages by increasing the antiviral innate immune response and interferon signalling. A total of 192 novel citrullination sites associated with inflammation, cell death and DNA/RNA processing pathways were identified in M1 and M2 macrophages. CONCLUSIONS: We showed that inhibiting PAD activity using a pharmacological inhibitor or silencing PAD2 with PAD2 siRNA shifted the activation of macrophages towards the M2 phenotype, which can be crucial for designing novel macrophage-mediated therapeutic strategies. We revealed a major citrullinated proteome and its rearrangement following macrophage polarization, which after further validation could lead to significant clinical benefits for the treatment of inflammation and autoimmune diseases.