Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response.

Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.

Phosphatidic acid binds to and stimulates the activity of ARGAH2 from Arabidopsis.

Phosphatidic acid (PA) has emerged as an important lipid signal during abiotic and biotic stress conditions such as drought, salinity, freezing, nutrient starvation, wounding and microbial elicitation. PA acts during stress responses primarily via binding and translocating target proteins or through modulating their activity. Owing to the importance of PA during stress signaling and developmental stages, it is imperative to identify PA interacting proteins and decipher their specific roles. In the present study, we have identified PA binding proteins from the leaves of Arabidopsis thaliana. Mass spectroscopy analysis led to the identification of 21 PA binding proteins with known roles in various cellular processes. One of the PA-binding proteins identified during this study, AtARGAH2, was further studied to unravel the role of PA interaction. Recombinant AtARGAH2 binding with immobilized PA on a solid support validated PA-AtARGAH2 binding invitro. PA binding to AtARGAH2 leads to the enhancement of arginase enzymatic activity in a dose dependent manner. Enzyme kinetics of recombinant AtARGAH2 demonstrated a lower Km value in presence of PA, suggesting role of PA in efficient enzyme-substrate binding. This simple approach could systematically be applied to perform an inclusive study on lipid binding proteins to elucidate their role in physiology of plants.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Gefitinib Results in Robust Host-Directed Immunity Against Salmonella Infection Through Proteo-Metabolomic Reprogramming.

The global rise of antibiotic-resistant strains of Salmonella has necessitated the development of alternative therapeutic strategies. Recent studies have shown that targeting host factors may provide an alternative approach for the treatment of intracellular pathogens. Host-directed therapy (HDT) modulates host cellular factors that are essential to support the replication of the intracellular pathogens. In the current study, we identified Gefitinib as a potential host directed therapeutic drug against Salmonella. Further, using the proteome analysis of Salmonella-infected macrophages, we identified EGFR, a host factor, promoting intracellular survival of Salmonella via mTOR-HIF-1α axis. Blocking of EGFR, mTOR or HIF-1α inhibits the intracellular survival of Salmonella within the macrophages and in mice. Global proteo-metabolomics profiling indicated the upregulation of host factors predominantly associated with ATP turn over, glycolysis, urea cycle, which ultimately promote the activation of EGFR-HIF1α signaling upon infection. Importantly, inhibition of EGFR and HIF1α restored both proteomics and metabolomics changes caused by Salmonella infection. Taken together, this study identifies Gefitinib as a host directed drug that holds potential translational values against Salmonella infection and might be useful for the treatment of other intracellular infections.

Role of VapBC12 Toxin-Antitoxin Locus in Cholesterol-Induced Mycobacterial Persistence.

The worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly nonreplicating and metabolically inert subset of the Mycobacterium tuberculosis population, called "persisters." The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a RNase toxin (VapC12) inhibits translation by targeting proT tRNA in M. tuberculosis This results in cholesterol-specific growth modulation that increases the frequency of generation of the persisters in a heterogeneous M. tuberculosis population. Also, a null mutant strain of this toxin (ΔvapC12) demonstrated an enhanced growth phenotype in a guinea pig model of M. tuberculosis infection, depicting its role in disease persistence. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin-antitoxin module in M. tuberculosis enhances persister formation during infection. The current findings provide an opportunity to target persisters, a new paradigm facilitating tuberculosis drug development.IMPORTANCE The current TB treatment regimen involves a combination of drugs administered for an extended duration that could last for 6 months to 2 years. This could lead to noncompliance and the emergence of newer drug resistance strains. It is widely perceived that the major culprits are the so-called nonreplicating and metabolically inactive "persister" bacteria. The importance of cholesterol utilization during the persistence stage of M. tuberculosis infection and its potential role in the generation of persisters is very intriguing. We explored the mechanism involved in the cholesterol-mediated generation of persisters in mycobacteria. In this study, we have identified a toxin-antitoxin (TA) system essential for the generation of persisters during M. tuberculosis infection. This study verified that M. tuberculosis strain devoid of the VapBC12 TA system failed to persist and showed a hypervirulent phenotype in a guinea pig infection model. Our studies indicate that the M. tuberculosis VapBC12 TA system acts as a molecular switch regulating persister generation during infection. VapBC12 TA system as a drug target offers opportunities to develop shorter and more effective treatment regimens against tuberculosis.

Proteome analysis revealed the essential functions of protein phosphatase PP2A in the induction of Th9 cells.

Proteomic analysis identifies post-translational functions of proteins, which remains obscure in transcriptomics. Given the important functions of Th9 cells in anti-tumor immunity, we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we performed a comprehensive proteomic analysis of murine Th0 and Th9 cells, and identified proteins that are enriched in Th9 cells. Pathway analysis identified an abundance of phosphoproteins in the proteome of Th9 cells as compared to Th0 cells. Among upregulated phosphoproteins, Ppp2ca (catalytic subunit of protein phosphatase, PP2A) was found to be highly enriched in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1, Th2, Th17 and Tregs, its role in the differentiation and functions of Th9 cells is not identified yet. Here we found that PP2A is required for the induction of Th9 cells, as PP2A inhibition leads to the suppression of IL-9 and expression of key transcription factors of Th9 cells. PP2A inhibition abrogates Th9 cell-mediated anti-tumor immune response in B16-OVA melanoma tumor model. Thus, we report that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells.

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Mycobacteria Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Mycobacterium tuberculosis Pathogenesis.

Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-ß, IL-1α, and IL-1ß levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013171.

Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells.

Zinc is an essential micronutrient which regulates diverse physiological functions and has been shown to play a crucial role in viral infections. Zinc has a necessary role in the replication of many viruses, however, antiviral action of zinc has also been demonstrated in in vitro infection models most likely through induction of host antiviral responses. Therefore, depending on the host machinery that the virus employs at different stages of infection, zinc may either facilitate, or inhibit virus infection. In this study, we show that zinc plays divergent roles in rotavirus and dengue virus infections in epithelial cells. Dengue virus infection did not perturb the epithelial barrier functions despite the release of virus from the basolateral surface whereas rotavirus infection led to disruption of epithelial junctions. In rotavirus infection, zinc supplementation post-infection did not block barrier disruption suggesting that zinc does not affect rotavirus life-cycle or protects epithelial barriers post-infection suggesting the involvement of cellular pathways in the beneficial effect of zinc supplementation in enteric infections. Zinc depletion by N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibited dengue virus and Japanese encephalitis virus (JEV) infection but had no effect on rotavirus. Time-of-addition experiments suggested that zinc chelation affected both early and late stages of dengue virus infectious cycle and zinc chelation abrogated dengue virus RNA replication. We show that transient zinc chelation induces ER stress and antiviral response by activating NF-kappaB leading to induction of interferon signaling. These results suggest that modulation of zinc homeostasis during virus infection could be a component of host antiviral response and altering zinc homeostasis may act as a potent antiviral strategy against flaviviruses.

Comparative analysis of homologous aminopeptidase PepN from pathogenic and non-pathogenic mycobacteria reveals divergent traits.

Mycobacterium tuberculosis (Mtb) secretes proteases and peptidases to subjugate its host. Out of its sixty plus proteases, atleast three are reported to reach host macrophages. In this study, we show that Mtb also delivers a lysyl alanine aminopeptidase, PepN (Rv2467) into host macrophage cytosol. Our comparative in silico analysis shows PepNMtb highly conserved across all pathogenic mycobacteria. Non-pathogenic mycobacteria including M. smegmatis (Msm) also encode pepN. PepN protein levels in both Mtb (pathogenic) and Msm (non-pathogenic) remain uniform across all in vitro growth phases. Despite such tight maintenance of PepNs' steady state levels, upon supplementation, Mtb alone allows accumulation of any excessive PepN. In contrast, Msm does not. It not only proteolyzes, but also secretes out the excessive PepN, be it native or foreign. Interestingly, while PepNMtb is required for modulating virulence in vivo, PepNMsm is essential for Msm growth in vitro. Despite such essentiality difference, both PepNMtb and PepNMsm harbor almost identical N-terminal M1-type peptidase domains that significantly align in their amino acid sequences and overlap in their secondary structures. Their C-terminal ERAP1_C-like domains however align much more moderately. Our in vitro macrophage-based infection experiments with MtbΔpepN-expressing pepNMsm reveals PepNMsm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepNMtb and PepNMsm interactomes and found them to barely coincide. While PepNMtb chiefly interacts with Mtb's secreted proteins, PepNMsm primarily coimmunoprecipitates with Msm's housekeeping proteins. Thus, despite high sequence homology and several common properties, our comparative analytical study reveals host-centric traits of pathogenic and bacterial-centric traits of non-pathogenic PepNs.

Differential Metabolic Pathway Analysis of the Proteomes of Leishmania donovani and Leptomonas seymouri.

PURPOSE: Although in trypanosomatids, monoxeny (Leptomonas) is ancestral to dixeny (Leishmania), however clinical cases of visceral leishmanisis with Leptomonas co-infection are increasingly being reported from India. Using a proteogenomic approach, a detailed proteome analysis of these two kinetoplastid parasites viz., Leishmania and its sister Leptomonas, to catalog the key proteins associated with and therefore possibly responsible for phenotype changes in Leptomonas evolution and domestication as co-infection with Leishmania is carried out. EXPERIMENTAL DESIGN: LC-MS/MS is utilized for this proteomic purpose. One Leishmania donovani WHO reference strain and two Leptomonas seymouri isolates, which are originally isolated from clinical cases of kala azar patients with different inherent drug sensitivity viz., responsive and unresponsive, are used in this study. RESULTS: A network analysis, leveraging protein-protein interaction data helped to find the roles of the proteins in carbon metabolism and biosynthesis of secondary metabolites which is seen to be altered under stress conditions like drug resistance. CONCLUSIONS AND CLINICAL RELEVANCE: The information provided about the metabolic pathways modulated when contrasting these two phenotypes may lead to the development of new strategies to block parasite differentiation within the host and to also circumvent the problem of drug resistance. This proteomic study also offers new grounds for the investigation of novel hypothetical proteins potentially playing a role in evolutionary biology the knowledge of which is essential for treatment of patients co-infected with these two kinetoplastid parasites.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher Vmax and lower Km than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw.

Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-ß-glucanase, ß-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers.

Synovial fluid proteome in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoinflammatory disorder that affects small joints. Despite intense efforts, there are currently no definitive markers for early diagnosis of RA and for monitoring the progression of this disease, though some of the markers like anti CCP antibodies and anti vimentin antibodies are promising. We sought to catalogue the proteins present in the synovial fluid of patients with RA. It was done with the aim of identifying newer biomarkers, if any, that might prove promising in future. METHODS: To enrich the low abundance proteins, we undertook two approaches-multiple affinity removal system (MARS14) to deplete some of the most abundant proteins and lectin affinity chromatography for enrichment of glycoproteins. The peptides were analyzed by LC-MS/MS on a high resolution Fourier transform mass spectrometer. RESULTS: This effort was the first total profiling of the synovial fluid proteome in RA that led to identification of 956 proteins. From the list, we identified a number of functionally significant proteins including vascular cell adhesion molecule-1, S100 proteins, AXL receptor protein tyrosine kinase, macrophage colony stimulating factor (M-CSF), programmed cell death ligand 2 (PDCD1LG2), TNF receptor 2, (TNFRSF1B) and many novel proteins including hyaluronan-binding protein 2, semaphorin 4A (SEMA4D) and osteoclast stimulating factor 1. Overall, our findings illustrate the complex and dynamic nature of RA in which multiple pathways seems to be participating actively. CONCLUSIONS: The use of high resolution mass spectrometry thus, enabled identification of proteins which might be critical to the progression of RA.

Proteomics of human aqueous humor.

The aqueous humor is a colorless, transparent fluid that fills the anterior chamber of the eye. It plays an important role in maintaining the intraocular pressure and providing nourishment to the lens and cornea. The constitution of the aqueous humor is controlled by the blood-aqueous barrier. Though this ocular fluid has been extensively studied, its role in ocular physiology is still not completely understood. In this study, aqueous humor samples were collected from 250 patients undergoing cataract surgery, subjected to multiple fractionation strategies and analyzed on a Fourier transform LTQ-Orbitrap Velos mass spectrometer. In all, we identified 763 proteins, of which 386 have been identified for the first time in this study. Sorbitol dehydrogenase (SORD), filensin (BFSP1), and phakinin (BFSP2) are some of the proteins that have not been previously reported in the aqueous humor. Gene Ontology analysis revealed 35% of the identified proteins to be extracellular, with a majority of them involved in cell communication and signal transduction. This study comprehensively reports 386 novel proteins that have important potential as biomarker candidates for future research into personalized medicine and diagnostics aimed towards improving visual health.

Proteomic analysis of human vitreous humor.

BACKGROUND: The vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing metabolic nutrient requirements of the lens, coordinating eye growth and providing support to the retina. It is in close proximity to the retina and reflects many of the changes occurring in this tissue. The biochemical changes occurring in the vitreous could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. In this study, we investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry. RESULTS: The vitreous humor was subjected to multiple fractionation techniques followed by LC-MS/MS analysis. We identified 1,205 proteins, 682 of which have not been described previously in the vitreous humor. Most proteins were localized to the extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). Classification based on molecular function showed that 27% had catalytic activity, 10% structural activity, 10% binding activity, 4% cell and 4% transporter activity. Categorization for biological processes showed 28% participate in metabolism, 20% in cell communication and 13% in cell growth. The data have been deposited to the ProteomeXchange with identifier PXD000957. CONCLUSION: This large catalog of vitreous proteins should facilitate biomedical research into pathological conditions of the eye including diabetic retinopathy, retinal detachment and cataract.

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Proteomic analysis of human osteoarthritis synovial fluid.

BACKGROUND: Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. RESULTS: We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. CONCLUSIONS: We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.

A multilectin affinity approach for comparative glycoprotein profiling of rheumatoid arthritis and spondyloarthropathy.

BACKGROUND: Arthritis refers to inflammation of joints and includes common disorders such as rheumatoid arthritis (RA) and spondyloarthropathies (SpAs). These diseases differ mainly in terms of their clinical manifestations and the underlying pathogenesis. Glycoproteins in synovial fluid might reflect the disease activity status in the joints affected by arthritis; yet they have not been systematically studied previously. Although markers have been described for assisting in the diagnosis of RA, there are currently no known biomarkers for SpA. MATERIALS AND METHODS: We sought to determine the relative abundance of glycoproteins in RA and SpA by lectin affinity chromatography coupled to iTRAQ labeling and LC-MS/MS analysis. We also used ELISA to validate the overexpression of VCAM-1, one of the candidate proteins identified in this study, in synovial fluid from RA patients. RESULTS AND DISCUSSION: We identified proteins that were previously reported to be overexpressed in RA including metalloproteinase inhibitor 1 (TIMP1), myeloperoxidase (MPO) and several S100 proteins. In addition, we discovered several novel candidates that were overexpressed in SpA including Apolipoproteins C-II and C-III and the SUN domain-containing protein 3 (SUN3). Novel molecules found overexpressed in RA included extracellular matrix protein 1 (ECM1) and lumican (LUM). We validated one of the candidate biomarkers, vascular cell adhesion molecule 1 (VCAM1), in 20 RA and SpA samples using ELISA and confirmed its overexpression in RA (p-value <0.01). Our quantitative glycoproteomic approach to study arthritic disorders should open up new avenues for additional proteomics-based discovery studies in rheumatological disorders.