Proteomic Analysis of Circulating IgA1-Containing Immune Complexes in Patients with IgA Nephropathy

IgA1-containing immune complexes (IgA1-IC) consisting of galactose-deficient IgA1 (Gal-deficient IgA1) and IgG autoantibodies are central to the pathogenesis of IgA nephropathy (IgAN). These IgA1-IC form in circulation, and additional proteins may be added before the deposition in the glomeruli. However, the composition of these circulating IgA1-IC is not fully understood. To address this gap in knowledge, we developed a novel proteomic workflow.

IgA1-IC from sera of patients with IgAN and healthy controls (HCs) were isolated by lectin-affinity chromatography followed by size-exclusion chromatography (SEC). IgA1 yield and molecular integrity were assessed by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, IgA1-IC samples were analyzed by using liquid chromatography-mass spectrometry (LC-MS). LC-MS results were analyzed by label-free quantification (LFQ) to identify proteins in IgA1-IC of patients with IgAN versus HCs versus proteomes of monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1).

We ascertained that 22 proteins were more abundant in the chromatographic fractions with IgA1-IC from patients with IgAN compared with similar fractions from HCs and other, uncomplexed IgA1 molecular forms. These proteins encompassed immunoglobulins, several complement proteins, including those with regulatory functions, and apolipoproteins. In addition, the identified proteins were validated by use of orthogonal serum preparation and analysis of previously published proteomics data.

Our new workflow better characterized the serum IgA1-IC of patients with IgAN by identifying proteins with elevated abundance in IgA1-IC in patients with IgAN, including complement components and apolipoproteins. Complement proteins identified in this study indicate that the alternative and lectin pathways are involved, but not the classical pathway.