Abstract
Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.
Highlights
From the ‡Biodesign Institute, Center for Personalized Diagnostics, Arizona State University, Tempe, Arizona 85287; §Engineering Arts LLC, Phoenix, Arizona 85076; ¶Benaroya Research Institute, Seattle, Washington 98101
Their discoveries often relied on Western blots using either serum samples or cell lysates followed by separation by either one dimensional (1D) or 2D gel electrophoresis followed by the identification/characterization of reactive protein bands/spots using mass spectrometry [7,8,9,10,11,12]
Concept of Contra Capture Protein Array—Our contra capture protein array was built on nucleic acidprogrammable protein array (NAPPA) platform that enables the display of thousands of full-length human proteins without the need for labor-intensive protein expression and purification [41]
Summary
Plasmid DNA was prepared and mixed with NAPPA printing buffer prior to printing as previously described [41] These expression vectors allow the in vitro expression of proteins of interest with a terminal HaloTag. C-terminal deletion mutants for both myelin basic protein (MBP) isoforms and fibrinogen alpha chain (FGA) were designed based on the following rule: if two arginines were separated by fewer than 9 amino acids, they were treated as a unit; if two arginines were separated by more than 9 amino acids, the amino acids in the middle was used to divide the two arginines into two mutants (supplemental Table S2). Absorbance at 450 nm was read on Perkin Elmer EnVision Multilabel Reader
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