Immunological Enrichment of Low-Abundance Proteins for Comparative Proteomics

In the last 20 years, proteomic studies have not yielded any FDA-approved protein antigen biomarkers for diseases. Commonly used antigen discovery method called Serological Proteome Analysis (SERPA), is not useful in identifying low abundance proteins. On the contrary, Antibody mediated identification of antigens (AMIDA) enriches low abundance protein targets and we believe could improve the discovery rate of true antigen biomarkers using proteomics. However, AMIDA has not been popular due to technical challenges. A major limitation is the contamination posed by antibodies that are used for the isolation of antigens. Antibodies being in high abundance, mask the signal of protein antigens and obstructs the mass spectrometry identification of antigens during the discovery phase of autoantigen biomarker screening. In an effort to improve the discovery of protein antigens, we present here a solution using a reversible protein capture reagent, called Biotin-CDM. Biotin-CDM can be incorporated into AMIDA in order to remove contaminating antibodies and enrich low abundance protein antigens. We use Biotin-CDM to reversibly tag all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2DE. Here, we describe the synthesis of Biotin-CDM and optimization of conditions to label proteins without affecting antibody-antigen interactions. We have successfully incorporated Biotin-CDM in AMIDA to identify antigens targeted by antibodies from Rheumatoid Arthritis (RA) and Interstitial lung disease associated with RA (RA-ILD) patients. Genetic predisposition and cigarette smoking have been linked to a post-translational modification called “citrullination/deimination” that is involved in generating antigens which trigger the formation of neo-epitopes (autoantibodies) in Rheumatoid Arthritis. To understand the pathogenesis of RA-ILD, we screened in vitro deiminated antigens that may be preferentially targeted by antibodies from RA vs RA-ILD patients. We identified several antigens including catalase and cAMP-specific 3',5'-cyclic phosphodiesterase 4D isoform PDE4D5 that immunoprecipitated with antibodies from RA & RA-ILD patients. Surprisingly, the same proteins immunoprecipitated in the treated and deiminated samples. The major changes were in the post-translational modifications (PTM) of the immunoprecipitated proteins. Further exploration into the PTM preference by RA/RA-ILD patient antibodies will need to be done in the future. Finally, we have performed comparative proteomics study on mitochondrial proteomes from Huntington’s disease cell line model. Here we show the benefits of using antibodies for the isolation of mitochondria and the critical importance of sample preparation for comparative proteomics.</p

A New method to help the discovery of diagnostic biomarkers for autoimmune diseases

<p>Department of Biological Sciences</p

Citrullinated Autoantigen Targets as Markers of Extra-Articular Disease in Rheumatoid Arthritis

Citrullination represents an increasingly recognized posttranslational modification stemming from underlying physiological stressors that dysregulate intracellular calcium flux. Although this enzymatic process mediated by various isoforms of peptidylarginine deiminase (PAD) is fairly ubiquitous (occurring in normal as well as pathological states), the immune response to citrullinated proteins is heavily influenced by underlying HLA status and therefore highly associated with rheumatoid arthritis (RA) (Szodoray et al. 2010). Given that the humoral immune responses to citrullinated proteins may serve as an immunological “fingerprint” in RA, the question is whether delineating targets of anti-citrullinated protein antibodies (ACPAs) can provide insight regarding the site where immune tolerance is bypassed/broken or clarify the underlying pathophysiology of articular and extra-articular manifestations in this systemic autoimmune disease—even if the relative contribution of protein deimination versus citrulline-targeted immunity remains unresolved. In fact, extensive investigation over the last 10–15 years has yielded an expanded repertoire of ACPA specificities potentially linked with defined extra-articular manifestations, such as premature atherosclerosis, myocardial dysfunction, and interstitial lung disease (ILD), which negatively impact clinical outcome. Fueling these discoveries, novel approaches for identifying citrullinated autoantigen/autoantibody combinations have supported the search for additional biomarkers of extra-articular involvement that should further elucidate the immunobiology of relevant systemic disease pathways in RA

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

AbstractProteomics technologies are often used for the identification of protein targets of the immune system. Here, we discuss the immunoproteomics technologies used for the discovery of autoantigens in autoimmune diseases where immune system dysregulation plays a central role in disease onset and progression. These autoantigens and associated autoantibodies can be used as potential biomarkers for disease diagnostics, prognostics and predicting/monitoring drug responsiveness (theranostics). Here, we compare a variety of methods such as mass spectrometry (MS)-based [serological proteome analysis (SERPA), antibody mediated identification of antigens (AMIDA), circulating immune complexome (CIC) analysis, surface enhanced laser desorption/ionization-time of flight (SELDI-TOF)], nucleic acid based serological analysis of antigens by recombinant cDNA expression cloning (SEREX), phage immunoprecipitation sequencing (PhIP-seq) and array-based immunoscreening (proteomic microarrays), luciferase immunoprecipitation systems (LIPS), nucleic acid programmable protein array (NAPPA) methods. We also review the relevance of immunoproteomic data generated in the last 10 years, with a focus on the aforementioned MS based methods

Recent Progress in the Development of Advanced Functionalized Electrodes for Oxygen Evolution Reaction: An Overview

Presently, the global energy demand for increasing clean and green energy consumption lies in the development of low-cost, sustainable, economically viable and eco-friendly natured electrochemical conversion process, which is a significant advancement in different morphological types of advanced electrocatalysts to promote their electrocatalytic properties. Herein, we overviewed the recent advancements in oxygen evolution reactions (OERs), including easy electrode fabrication and significant action in water-splitting devices. To date, various synthetic approaches and modern characterization techniques have effectively been anticipated for upgraded OER activity. Moreover, the discussed electrode catalysts have emerged as the most hopeful constituents and received massive appreciation in OER with low overpotential and long-term cyclic stability. This review article broadly confers the recent progress research in OER, the general mechanistic approaches, challenges to enhance the catalytic performances and future directions for the scientific community

Recent Advances in Nanoscale Based Electrocatalysts for Metal-Air Battery, Fuel Cell and Water-Splitting Applications: An Overview

Metal-air batteries and fuel cells are considered the most promising highly efficient energy storage systems because they possess long life cycles, high carbon monoxide (CO) tolerance, and low fuel crossover ability. The use of energy storage technology in the transport segment holds great promise for producing green and clean energy with lesser greenhouse gas (GHG) emissions. In recent years, nanoscale based electrocatalysts have shown remarkable electrocatalytic performance towards the construction of sustainable energy-related devices/applications, including fuel cells, metal-air battery and water-splitting processes. This review summarises the recent advancement in the development of nanoscale-based electrocatalysts and their energy-related electrocatalytic applications. Further, we focus on different synthetic approaches employed to fabricate the nanomaterial catalysts and also their size, shape and morphological related electrocatalytic performances. Following this, we discuss the catalytic reaction mechanism of the electrochemical energy generation process, which provides close insight to develop a more efficient catalyst. Moreover, we outline the future perspectives and challenges pertaining to the development of highly efficient nanoscale-based electrocatalysts for green energy storage technology

In-gel equilibration for improved protein retention in 2DE-based proteomic workflows.

<p>The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second-dimension SDS-PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in-solution-based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second-dimension SDS-PAGE gel, referred to as in-gel equilibration. We show that in-gel equilibration is effective at reduction and alkylation in SDS-PAGE gels. Quantification of whole-cell extracts separated on 2DE gels shows that in-gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.</p

Developing Low-Cost, High Performance, Robust and Sustainable Perovskite Electrocatalytic Materials in the Electrochemical Sensors and Energy Sectors: “An Overview”

Since its discovery in 1839, research on the synthesis and application of perovskite materials has multiplied largely due to their suitability to be used in the fields of nanotechnology, chemistry and material science. Appropriate changes in composition or addition of other elements or blending with polymers may result in new hybrid and/or composite perovskite materials that will be applied in advanced fields. In this review, we have recapitulated the recent progress on perovskite nanomaterial in solar cell, battery, fuel cell and supercapacitor applications, and the prominence properties of perovskite materials, such as excellent electronic, physical, chemical and optical properties. We discussed in detail the synthesis and results of various perovskite hybrid nanomaterials published elsewhere. We have also discussed the results of various studies on these low dimensional composite nanomaterials in broad sectors such as electronics/optoelectronics, batteries, supercapacitors, solar cells and electrochemical sensors

Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins

Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics