A method to identify trace sulfated IgG N-glycans as biomarkers for rheumatoid arthritis

Abstract

N-linked glycans on immunoglobulin G (IgG) have been associated with pathogenesis of diseases and the therapeutic functions of antibody-based drugs; however, low-abundance species are difficult to detect. Here we show a glycomic approach to detect these species on human IgGs using a specialized microfluidic chip. We discover 20 sulfated and 4 acetylated N-glycans on IgGs. Using multiple reaction monitoring method, we precisely quantify these previously undetected low-abundance, trace and even ultra-trace N-glycans. From 277 patients with rheumatoid arthritis (RA) and 141 healthy individuals, we also identify N-glycan biomarkers for the classification of both rheumatoid factor (RF)-positive and negative RA patients, as well as anti-citrullinated protein antibodies (ACPA)-positive and negative RA patients. This approach may identify N-glycosylation-associated biomarkers for other autoimmune and infectious diseases and lead to the exploration of promising glycoforms for antibody therapeutics.

Variable domain N‐linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B‐cell selection

“…These data are in line with and extend previous observations of Fab glycosylation in polyclonal anti‐citrullinated protein responses in RA, non‐expressed BCR sequences from CCP‐sorted cells, and a limited number of expressed monoclonals . In the current study, we also found that ACPA Fab glycans in the recombinant IgG were distinctly different from the Fc glycans and highly sialylated, consistent with what has been determined for Fab glycans of serum polyclonal antibodies in RA patients . Notably, a subset of the investigated mAbs have recently been found to have substantial in vitro and in vivo functionality and mediate enhanced osteoclastogenesis, pain behavior and endotoxin‐induced arthritis .…”

“…The Fab glycopeptide profile of recombinantly expressed IgG from HEK‐293 cell systems cannot be expected to have the exact same glycan‐composition as what has been reported for PNGase F released Fab‐glycans analyzed from human polyclonal ACPA‐IgG . Yet, many of the complex sialylated forms identified herein have been detected in RA patients . In any case and more importantly, our results clearly indicate that the monoclonal IgG indeed have occupied high‐sialic acid Fab‐glycosylation sites.…”

Glycomic Signatures of Plasma IgG Improve Preoperative Prediction of the Invasiveness of Small Lung Nodules

“…There have been few reports about triand tetra-antennary structures on human IgG. However, with the development of sensitive detection technologies, increasing numbers of trace glycans, including the tri-and tetra-antennary structures, have been observed on serum IgG, some of which have been identified as novel biomarkers for disease [37]. Our study findings suggest the high sensitivity of the lectin microarray will be useful for the discovery of trace glycans.…”

Advances in mass spectrometry‐based glycomics

“…Conventionally, lectin‐based enrichment , hydrophilic interaction chromatography (HILIC) , porous graphitized carbon (PGC) and hydrazide based methods , are extensively used for the purification and enrichment of glycans. Over the past 5 years, several studies reported the use of a variety of materials to facilitate high‐throughput sample preparation .…”