Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

• Published in: Journal of proteome research 2013-12, Vol.12 (12), p.5656-5665
• Main Authors: Merl, Juliane, Deeg, Cornelia A, Swadzba, Margarete E, Ueffing, Marius, Hauck, Stefanie M
• Format: Article
• Language: English
• Subjects: Animals; Antigen-Antibody Complex - chemistry; Antigen-Antibody Complex - isolation & purification; Autoantibodies - chemistry; Autoantibodies - isolation & purification; Autoantigens - chemistry; Autoantigens - isolation & purification; Autoimmune Diseases; Chromatography, Liquid - methods; Extracellular Matrix Proteins - isolation & purification; Extracellular Matrix Proteins - metabolism; Horse Diseases - immunology; Horse Diseases - metabolism; Horse Diseases - pathology; Horses; Humans; Mass Spectrometry - methods; Matrix Metalloproteinase Inhibitors - isolation & purification; Matrix Metalloproteinase Inhibitors - metabolism; Molecular Sequence Annotation; Osteopontin - isolation & purification; Osteopontin - metabolism; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; T-Lymphocytes - pathology; Uveitis - immunology; Uveitis - metabolism; Uveitis - pathology; Uveitis - veterinary; Vitreous Body - chemistry; Vitreous Body - immunology; Vitreous Body - pathology
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr4005986

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future.