Profiling of Endo H‐released serum N‐glycans using CE‐LIF and MALDI‐TOF‐MS – Application to rheumatoid arthritis

Abstract

High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-β-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man(5-9)GlcNAc(1) represented the majority of the pool. The monoglucosylated structure Glc(1)Man(9)GlcNAc(1) as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of α-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and α-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans.

N-Glycosylation Profile of Undifferentiated and Adipogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells: Towards a Next Generation of Stem Cell Markers

“…The changes in relative amount of neutral hybrid-type N-glycans H5N2 (m/z 1579.7) and H6N2 (m/z 1783.8) in 15 days adipogenically differentiated MSCs and undifferentiated MSCs were statistically significant. In addition, a glycan of the composition H10N1 was detected in trace amounts at m/z 2355.1, which was verified as the monoglucosylated precursor H10N1 high-mannose-type Nglycan that has been previously reported to be present in human blood serum [36]. In general, the average relative amounts of Endo H-released N-glycans of undifferentiated and adipogenically differentiated MSCs (5 or 15 days) were more different than those of 5 and 15 days adipogenically differentiated MSCs ( Fig.…”

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS

“…Aberrant glycosylation happens on several important serum glycoproteins, such as IgG, haptoglobin (Hp), and alpha-1-acid glycoprotein (AGP) in RA patients [24–26]. Previous studies investigated the N-glycan patterns in the serum of RA patients [27–29]. However, the comparison of glycans between RA patients and healthy controls in detail has not yet been investigated, since the isomeric glycans were difficult to be identified and annotated.…”

Glycoproteomics on the rise: Established methods, advanced techniques, sophisticated biological applications

“…High‐mannose and hybrid‐type N‐ glycans with roles in immune response were released from human sera of rheumatoid arthritis patients (50 μL) with Endo‐H, separated from serum using C18‐cartridges (glycans in the flow‐through), fractionated on a high‐pH anion exchange column with pulsed amperometric detection, desalted and analyzed by MALDI‐TOF‐MS, or, labeled with APTS for CE‐LIF. The study found that high‐mannose structures Man 5–9 GlcNAc 1 were predominant, and one monoglucosylated structure Glc 1 Man 9 GlcNAc 1 and four hybrid structures were identified .…”