Background/Purpose: Anti-double-stranded (ds)DNA autoantibodies are prototypic serological markers of systemic lupus erythematosus (SLE) but little is known about their immunoglobulin variable (IgV) region composition at the level of the secreted (serum) proteome. Here, we use a novel proteomic workflow based on de novo database mass spectrometric sequencing of anti-dsDNA precipitins to analyse IgV subfamily expression and mutational signatures of high-affinity, precipitating anti-dsDNA responses.

Methods: Anti-dsDNA antibodies were purified from serum precipitins reactions prepared by agarose gel immunodiffusion between circular plasmid dsDNA and SLE serum from eight patients testing positive for anti-dsDNA by Farr radioimmunoassays (RIA). Microgram amounts of precipitating anti-dsDNA Igs were separated by SDS-PAGE, and in-gel tryptic and chymotryptic digests were performed on the heavy (H) and light (L)-chain bands and peptides were subjected to nano-high performance liquid chromatography-mass spectrometry followed by combined de novo amino acid sequencing and database matching using Peaks 8.0 software utilising ImMunoGeneTics (IMGT) and Uniprot databases. L-chain complementarity determining region 3 (CDR3) peptides were used as biomarkers to track serum anti-dsDNA clonotypes using quantitative multiple reaction monitoring (MRM).

Results: Serum anti-dsDNA proteomes were oligoclonal with shared (public) expression of IgG heavy chain variable region (IGHV) and kappa chain variable region (IGKV) subfamilies. IgV peptide maps from eight subjects showed extensive public and random (private) amino acid replacement mutations with prominent arginine substitutions across H- and L-chains. Shared sets of LCDR3 peptides specified by arginine substitutions were sequenced from the dominantly expressed IGKV3-20 subfamily, with changes in expression levels of a clonal L-chain CDR3 peptide by quantitative MRM paralleling the rise and fall of anti-dsDNA levels by Farr RIA. The heavily mutated IgV peptide signatures of precipitating anti-dsDNA autoantibody proteomes reflect the strong selective forces that shape humoral anti-dsDNA responses in germinal centres.

Conclusion: This first comprehensive proteomic analysis of precipitating anti-dsDNA IgV peptide repertoires in lupus serum offers a fresh approach for characterising and monitoring anti-dsDNA clonal populations with MS precision and accuracy, and for comparing their molecular signatures with other high-throughput “omics” technologies. Direct sequencing of agarose gel precipitins using microlitre volumes of stored sera streamlines the antibody sequencing workflow and is generalisable to other precipitating serum antibodies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/precipitating-anti-dsdna-peptide-repertoires-in-lupus/