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Patient‐specific Autoantigen Sample Preparation and Analysis Using the Reversible Protein Tag ProMTag
Author(s) -
Minden Jonathan S.,
Ascherman Dana,
Lucas Amber
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r6028
Subject(s) - autoantibody , immunoprecipitation , antibody , covalent bond , chemistry , antigen , autoimmune disease , autoimmunity , medicine , immunology , computational biology , microbiology and biotechnology , biochemistry , biology , organic chemistry
Autoimmune diseases affect >20 million people in the US today. Currently, disease‐specific autoantibodies are thought to be the best biomarkers for diagnosis. Conventional immunoprecipitation methods have been used to identify autoantigens from the most common autoimmune diseases. However, these diseases account for only 6.5 million of the 20 million patients suffering from autoimmune diseases, leaving many without diagnoses until irreversible damage occurs. The remaining 13.5 million patients have >70 autoimmune disorders without well characterized autoantibodies. The state‐of‐the‐art diagnostic test of these remaining diseases relies on gel electrophoresis of immunoprecipitated radiolabeled proteins, which cannot be identified by MS due to safety issues and the overwhelming presence of immunoglobulins. We have created an immunoprecipitation method that uses serum from patients with any autoimmune disorder to identify patient‐specific autoantigen proteins. This method uses a reversible click chemistry tag, called ProMTag. One end of the ProMTag forms a reversible, covalent bond with protein by coupling to lysines and amino termini. The other end of the ProMTag can form an irreversible, covalent bond with a solid bead support via a click chemistry, methyltetrazine‐TCO, pairing. In this study, the proteins of cell lysates that contain potential autoantigens were labeled with ProMTag. The ProMTagged‐proteins were exposed to patient antibodies bound to Protein A beads, thus capturing the ProMTagged autoantigens. All proteins were released from the Protein A beads, including ProMTagged‐autoantigens and untagged‐antibodies. The ProMTagged‐autoantigens were subsequently coupled to TCO beads, and the untagged‐antibodies were washed away. The linkage between the ProMTag and autoantigens was then reversed, yielding autoantigen proteins with greatly reduced antibody contamination ready for MS analysis. MS analysis successfully identified autoantigens from patient serums with rheumatoid arthri. This autoimmune biomarker discovery method can accelerate sample testing for known autoantigens and facilitating rapid discovery of novel autoantigens for both diagnostic and predictive biomarkers.