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.Post-translational modifications can affect antibody function in health and disease, but identification of all variants is difficult using existing technologies. Here the authors develop a microfluidic method to identify and quantify low-abundance IgG N-glycans and show some of these IgGs can be used as biomarkers for rheumatoid arthritis.

Fig. 1

On-chip enrichment of acidic N-glycans. a Diagram of the specialized TiO₂-PGC chip. b Extracted compound chromatograms (ECC) of N-glycans detected in the load, flow-through (neutral N-glycans) and eluate fractions (acidic N-glycans) of the TiO₂ enrichment column as analyzed by liquid chromatography coupled with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry in the positive mode. c TiO₂ enrichment column selectively captures acidic N-glycans. Neutral N-glycans (blue dot) do not efficiently bind to TiO₂ and are recovered in the flow-through fraction, whereas acidic N-glycans are retained on TiO₂ during the load and flow-through steps and are eluted with the elution buffer. The enrichment recovery rates (numbers beside the dots) of five acidic N-glycan standards (red dots with black circles) are also given

Fig. 2

The greatly improved detection of acidic N-glycans after enrichment on the TiO₂-PGC chip. In a, the [M+3H]³⁺ ions of an acidic N-glycan were overlapped with the isotopic ions of co-eluted neutral N-glycans and could not be assigned accurately. Using the TiO₂-PGC chip, the acidic N-glycans were enriched efficiently and detected with an enhanced S/N and improved mass accuracy because of the removal of interference from neutral N-glycans. In b, the signal intensity as well as the S/N of acidic N-glycans were significantly enhanced after on-chip enrichment because of the reduced ion-suppression derived from neutral N-glycans/matrix. In c, with the removal of noise derived from neutral N-glycans/matrix, the acidic N-glycan mass accuracy was significantly enhanced, thus providing reliable evidence for the identification of acidic N-glycans. The number in the bracket beside each mass value indicates the mass error (p.p.m.)

Fig. 3

Comprehensive profiling of N-glycans on serum IgGs. a The number of N-glycans characterized on serum IgGs using the TiO₂-PGC chip coupled with Q-TOF MS. b Structural maps of N-glycans on IgG. c–f MS/MS of representative sulfated N-glycans

Fig. 4

Structures of sulfated N-glycans identified on human serum IgGs. a Structures of identified sulfated N-glycans in human serum IgG. (*represents sulfated N-glycans that have been reported from a human source; ^#represents sulfated N-glycans that have been reported on porcine thyroglobulin; another 11 sulfated N-glycans have not been previously reported). b Four exoglycosidases, Sialidase C, β1–4 galactosidase, β-N-acetyl glucosaminidase, and α1–2, 3 mannosidase, were employed to hydrolyze the sulfated N-glycans to determine site of the sulfate group. c The retention times and peak patterns of sulfated N-glycans in human IgGs (red) were compared with those in porcine thyroglobulin (blue), which has been well characterized. d The retention time of each sulfated N-glycan (black) is 0.5 min later than its corresponding parent N-glycans (red), and the signal intensity is 3-fold to 200-fold lower than that of the non-sulfated N-glycans

Fig. 5

Improved detection of N-glycans using the MRM method. N-glycans were detected using Q-TOF MS (pale blue or pale red) and QQQ MS (MRM mode, blue or red). The improved detection of representative neutral N-glycans Hex₄HexNAc₄, Hex₄HexNAc₄dHex₁ and Hex₃HexNAc₅dHex₁ are shown in a, and the improved detection of representative sialylated N-glycans Hex₅HexNAc₃dHex₁NeuAc₁, Hex₅HexNAc₄dHex₁NeuAc₁ + SO₃ and Hex₆HexNAc₃NeuAc₁ are shown in b. The signal intensities of N-glycans obtained using the MRM method were increased by up to 180-fold compared with those obtained with Q-TOF MS. Notably, the S/N of the acidic N-glycans obtained using MRM increased by ~10-fold to 1000-fold compared with that obtained with Q-TOF MS

Fig. 6

Glycomic analysis of human serum IgGs using MRM method. It revealed a highly dynamic range of up to five orders of magnitude in the relative abundances of both neutral a and acidic b N-glycans, data are shown as mean ± s.d

Fig. 7

Performance and relative abundances of the potential N-glycan biomarkers for RA. a Symbols depicting N-glycan biomarkers identified in current study (SGm1 and SGm2) and that reported previously (G0/G1). b The ROC curve of the biomarkers for the classification of RA (n = 90) and healthy individuals (n = 57). The results are plotted for the training set. c, d The scatter plot of the relative abundance of the N-glycan biomarker in control individuals (training set: n = 57, validation set: n = 84), RF-positive [RA (RF+)] (training set: n = 71, validation set: n = 90) and RF-negative [RA (RF−)] RA patients (training set: n = 19, validation set: n = 97), as well as ACPA-positive [RA (ACPA+)] (training set: n = 75, validation set: n = 132) and ACPA-negative [RA (ACPA−)] RA patients (training set: n = 15, validation set: n = 55), in training set and validation set. e The scatter plot of the relative abundance of the N-glycan biomarkers in AS patients (n = 34), OA patients (n = 26) and in controls of each (AS: n = 27, OA: n = 45). Horizontal lines indicate the median. Statistical analysis was performed by non-parametric Mann–Whitney test, two-sided (*P < 0.05, **P < 0.01, ***P < 0.001)