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

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.