Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

Ganesan, Vinitha; Ascherman, Dana P.; Minden, Jonathan S.

doi:10.1515/bmc-2016-0007

Cited by 20 publications

(12 citation statements)

References 89 publications

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“…Immunoproteomic technologies are widely used to discover autoantigens and autoantibodies which may serve as prognostic, diagnostic and theranostic biomarkers [19]. Immunological assays like ELISA are considered the gold standard in clinical settings, and are advantageous due to higher throughput than mass spectrometric analyses [20].…”

Section: Discussionmentioning

confidence: 99%

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

et al. 2019

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Background Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn’s disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings. Electronic supplementary material The online version of this article (10.1186/s12014-019-9246-0) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

et al. 2019

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“…These targets await further study. It should be noted that each autoantigen discovery approach has its own characteristic strengths and weaknesses ( 89 ). Thus, the different strategies are best viewed as complementary rather than competing or redundant.…”

Section: Novel Technologies In T-cell Response and Autoantigen Identimentioning

confidence: 99%

The Evolving Landscape of Autoantigen Discovery and Characterization in Type 1 Diabetes

2019

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Type 1 diabetes (T1D) is an autoimmune disease that is caused, in part, by T cell–mediated destruction of insulin-producing β-cells. High risk for disease, in those with genetic susceptibility, is predicted by the presence of two or more autoantibodies against insulin, the 65-kDa form of glutamic acid decarboxylase (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8). Despite this knowledge, we still do not know what leads to the breakdown of tolerance to these autoantigens, and we have an incomplete understanding of T1D etiology and pathophysiology. Several new autoantibodies have recently been discovered using innovative technologies, but neither their potential utility in monitoring disease development and treatment nor their role in the pathophysiology and etiology of T1D has been explored. Moreover, neoantigen generation (through posttranslational modification, the formation of hybrid peptides containing two distinct regions of an antigen or antigens, alternative open reading frame usage, and translation of RNA splicing variants) has been reported, and autoreactive T cells that target these neoantigens have been identified. Collectively, these new studies provide a conceptual framework to understand the breakdown of self-tolerance, if such modifications occur in a tissue- or disease-specific context. A recent workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases brought together investigators who are using new methods and technologies to identify autoantigens and characterize immune responses toward these proteins. Researchers with diverse expertise shared ideas and identified resources to accelerate antigen discovery and the detection of autoimmune responses in T1D. The application of this knowledge will direct strategies for the identification of improved biomarkers for disease progression and treatment response monitoring and, ultimately, will form the foundation for novel antigen-specific therapeutics. This Perspective highlights the key issues that were addressed at the workshop and identifies areas for future investigation.

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“…However, Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation http://dx.doi.org/10.5772/intechopen.75200 the inherently high gel-to-gel variability and relatively low resolving power of individual gels impacts on the accuracy of spot picking and imposes a limitation due to co-migrating proteins, which is especially problematic for low-abundance protein targets. Several modifications have been suggested to address such limitations, including multi-colour fluorescence-based 2-D gel immunoproteomics approaches [62], but these still do not address the fundamental issues of the limited resolving power of the gels, the modest limit of detection or the throughput for SERPA. This technology is thus less widely used for autoantigen discovery now and has been largely supplanted by newer technologies that are better able to overcome these limitations.…”

Section: Serologic Proteome Analysismentioning

confidence: 99%

“…SEREX has also been successfully used to identify several TAAs that generate a humoral immune response in cancers such as those from the kidney, lung, breast and colon [63]. However, a fundamental limitation of SEREX is that the method lacks the ability to differentiate or detect post-translational modifications (PTMs) that are likely to play a significant role in autoimmune diseases [62] and cancers [63]. This approach also restricts the types of TAAs identified to those that can be expressed in a prokaryotic system and also effectively excludes TAAs that require folding mechanisms unique to eukaryotes to achieve the correct conformational epitope for recognition [63].…”

Section: Serological Analysis Of Recombinant Cdna Expression Librariementioning

confidence: 99%

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

Aziz¹,

Smith²,

Blackburn³

2019

Autoantibodies and Cytokines

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Autoantibodies produced against self-antigens, or 'autoantigens', result from a loss of selftolerance triggered by genetic and/or environmental factors which induce the immune system to attack the host's own cells, resulting in a condition referred to as autoimmunity. In classic autoimmune diseases, it is well established that the pathology relates directly to the autoantibodies. However, it is increasingly recognised that autoantibodies are also found in many other disease areas, including cancers, cardiovascular and neurodegenerative diseases, as well infectious diseases such as malaria, albeit in such diseases it is unclear whether the autoantibodies play a direct role in the pathology or whether they are merely symptomatic of disease. Irrespective of whether they are causative or symptomatic of specific diseases though, there is increasing interest globally in exploring the clinical potential of circulating autoantibodies as diagnostic biomarkers. This chapter provides an overview of the diagnostic utility of autoantibody biomarkers in a range of disease areas and discusses their potential utility in disease staging, treatment monitoring and in prediction of immune-related adverse events. It also provides an overview of traditional and contemporary technological approaches to autoantibody biomarker discovery and validation, focusing on protein microarrays that are ideally suited to this important area of research.

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Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

Cited by 20 publications

References 89 publications

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

The Evolving Landscape of Autoantigen Discovery and Characterization in Type 1 Diabetes

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

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