Deep coverage and quantification of the bone proteome provides enhanced opportunities for new discoveries in skeletal biology and disease.

Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with ≥ 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Component of the Senescence-Associated Secretory Phenotype.

Cellular senescence is a stress or damage response by which a cell adopts of state of essentially permanent proliferative arrest, coupled to the secretion of a number of biologically active molecules. This senescence-associated secretory phenotype (SASP) underlies many of the degenerative and regenerative aspects of cellular senescence - including promoting wound healing and development, but also driving diabetes and multiple age-associated diseases. We find that nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in nicotinamide adenine dinucleotide (NAD) biosynthesis, is elevated in senescent cells without a commensurate increase in NAD levels. This elevation is distinct from the acute DNA damage response, in which NAD is depleted, and recovery of NAD by NAMPT elevation is AMPK-activated protein kinase (AMPK)-dependent. Instead, we find that senescent cells release extracellular NAMPT (eNAMPT) as part of the SASP. eNAMPT has been reported to be released as a catalytically active extracellular vesicle-contained dimer that promotes NAD increases in other cells and extends lifespan, and also as free monomer that acts as a damage-associated molecular pattern and promotes conditions such as diabetes and fibrosis. Senescent cells released eNAMPT as dimer, but surprisingly eNAMPT appeared in the soluble secretome while being depleted from exosomes. Finally, diabetic mice showed elevated levels of eNAMPT, and this was lowered by treatment with the senolytic drug, ABT-263. Together, these data reveal a new SASP factor with implications for NAD metabolism.

Rapid Discrimination of Malaria- and Dengue-Infected Patients Sera Using Raman Spectroscopy.

Malaria and dengue have overlapping clinical symptoms and are prevalent in the same geographic region (tropical and subtropical), hence precise diagnosis is challenging. The high mortality rate associated with both malaria and dengue could be attributed to "false", "delayed", or "missed" diagnosis. The present study thus aims to stratify malaria and dengue using Raman spectroscopy (RS). In total, 130 human sera were analyzed for model development and double-blinded testing. Principal components linear discriminant analysis (PC-LDA) of acquired RS-spectra could classify malaria and dengue with a minor overlap of 16.7%. Receiver operating characteristic (ROC) analysis of test samples showed sensitivity/specificity of 0.9529 for malaria vs healthy controls (HC) and 0.9584 for dengue vs HC. The Raman findings were complemented by mass spectroscopy (MS)-based metabolite analysis of 8 individuals, each from malaria, dengue, and HC. Several of the metabolites, including amino acids, cell-free DNA, creatinine, and bilirubin, assigned for the predominant RS-bands were also identified by MS and showed similar trends. Our data clearly indicates that RS-based serum analysis using a microprobe has immense potential for early, accurate, and automated detection and discrimination of malaria and dengue, and in the future, it could be extrapolated in field-settings combined with hand-held RS. Further, this approach might be extended to diagnose other closely related infections with similar clinical manifestations.

Clinical Proteomics and Cytokine Profiling for Dengue Fever Disease Severity Biomarkers.

Dengue fever (DF) is a major global health burden with a pathophysiology that is still incompletely understood. Biomarkers that predict and explain susceptibility to DF and its progression to its more severe hemorrhagic form are much needed. DF is endemic in tropical and subtropical regions of the world, with a rapidly increasing incidence of disease severity. We conducted a clinical biomarker discovery study using both a case-control and longitudinal study design. Plasma proteome alterations in patients with DF (n = 12) and dengue hemorrhagic fever (DHF, n = 24) were analyzed in comparison to healthy controls (HCs, n = 16), using the isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics methodology (false discovery rate of 1%, ≥2 peptides). Several proteins such as the alpha-2 macroglobulin, angiotensinogen, apolipoprotein B-100, serotransferrin, and ceruloplasmin were upregulated (fold change >1.2) in all DHF cases, and downregulated in DF (fold change <0.83), compared with HCs. Plasma cytokine profiling (8 DF, 8 DHF, and 8 HC) on two consecutive time points, at day 0 (day of admission) and days 5-7, found significant elevation in IL-1RA, IL-7, TNF-α, MCP1-MCAF, and MIP-1ß levels, but only in the DHF cases, which is the severe disease, and not in DF, compared with HCs (p < 0.05). These new observations on changes in the plasma proteome and cytokine profiles in patients with dengue infection identify several putative molecular leads for future biomarker development and precision medicine in relation to forecasting DF disease severity.

Quantitative Proteomics Analysis of Plasmodium vivax Induced Alterations in Human Serum during the Acute and Convalescent Phases of Infection.

The radial distribution of Plasmodium vivax malaria burden has evoked enormous concern among the global research community. In this study, we have investigated the serum proteome alterations in non-severe vivax malaria patients before and during patient recuperation starting from the early febrile to the defervescence and convalescent stages of the infection. We have also performed an extensive quantitative proteomics analysis to compare the serum proteome profiles of vivax malaria patients with low (LPVM) and moderately-high (MPVM) parasitemia with healthy community controls. Interestingly, some of the serum proteins such as Serum amyloid A, Apolipoprotein A1, C-reactive protein, Titin and Haptoglobin, were found to be sequentially altered with respect to increased parasite counts. Analysis of a longitudinal cohort of malaria patients indicated reversible alterations in serum levels of some proteins such as Haptoglobin, Apolipoprotein E, Apolipoprotein A1, Carbonic anhydrase 1, and Hemoglobin subunit alpha upon treatment; however, the levels of a few other proteins did not return to the baseline even during the convalescent phase of the infection. Here we present the first comprehensive serum proteomics analysis of vivax malaria patients with different levels of parasitemia and during the acute and convalescent phases of the infection.

Proteomics of Plasmodium vivax malaria: new insights, progress and potential.

INTRODUCTION: Plasmodium vivax has accounted for an enormous share of the global malaria burden in recent years, along with Plasmodium falciparum. The wide distribution of P. vivax and recent evidences of severe and complicated vivax malaria across several endemic regions of the world suggest that this disease may have been more overlooked than benign. While P. falciparum has been extensively studied, P. vivax has received limited research attention owing to its complex nature and absence of a continuous culture system. AREAS COVERED: This review briefly describes the epidemiology of vivax malaria, analyzes challenges towards effective control and summarizes major insights provided by genomics and transcriptomics research in the area. Subsequently, the review provides a detailed description of the applications of proteomics in vivax malaria research, focusing on both host responses and parasite proteomics studies to understand P. vivax biology. Expert commentary: In recent years, proteomics technologies are being used effectively to understand P. vivax biology and the underlying pathogenesis. Technological advances in mass spectrometry configurations, multiomics investigations and emerging strategies such as targeted proteomics may also immensely aid in studying disease severity, improving existing diagnosis and identifying new drug and vaccine targets.

Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria.

In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.

Metabolomics and its integration with systems biology: PSI 2014 conference panel discussion report.

Metabolomics, being a relatively new field, is facing multiple challenges related to data acquisition and interpretation, reproducibility across analytical platforms, integration with other omics approaches and translation into theragnostic biomarkers. There is an immediate need to overcome these challenges in order to make metabolomics more useful and reliable in terms of improving our current understanding of disease biology and help in developing predictive biomarkers. Researchers interested in metabolomics gathered for a panel discussion on 'Metabolomics and its integration with systems biology' during the 6th Annual Meeting of Proteomics Society-India and International Conference on "Proteomics from Discovery to Function" held at the Indian Institute of Technology, Bombay from December 7-9, 2014. The panel discussed various challenges related to metabolomics and also proposed several effective solutions for optimum implementation of metabolomics in clinical practice. The key areas of panel discussion were improvement in metabolite databases with comprehensive spectral libraries, need for extensive bioinformatics tools for integrative approaches and serious considerations for clinical validation of the biomarkers for the successful implementation of metabolomics in clinics. BIOLOGICAL SIGNIFICANCE: Information drafted in this report is significant for researchers working in metabolomics field to overcome the challenges and successful implementation of metabolomics in clinical practice. This article is part of a special issue titled: Proteomics in India.

Differential expression of serum/plasma proteins in various infectious diseases: specific or nonspecific signatures.

Apart from direct detection of the infecting organisms or biomarker of the pathogen itself, surrogate host markers are also useful for sensitive and early diagnosis of pathogenic infections. Early detection of pathogenic infections, discrimination among closely related diseases with overlapping clinical manifestations, and monitoring of disease progression can be achieved by analyzing blood biomarkers. Therefore, over the last decade large numbers of proteomics studies have been conducted to identify differentially expressed human serum/plasma proteins in different infectious diseases with the intent of discovering novel potential diagnostic/prognostic biomarkers. However, in-depth review of the literature indicates that many reported biomarkers are altered in the same way in multiple infectious diseases, regardless of the type of infection. This might be a consequence of generic acute phase reactions, while the uniquely modulated candidates in different pathogenic infections could be indicators of some specific responses. In this review article, we will provide a comprehensive analysis of differentially expressed serum/plasma proteins in various infectious diseases and categorize the protein markers associated with generic or specific responses. The challenges associated with the discovery, validation, and translational phases of serum/plasma biomarker establishment are also discussed.