Rheumatoid arthritis (RA) is the most common inflammatory polyarthritis. In addition, 60–80% of patients express anti-citrullinated protein antibodies (ACPAs), which serve as a diagnostic marker for RA. The effector functions of these autoantibodies can be heavily affected by the N-glycosylation of their Fc region. Here we present a comparison of the Fc N-glycosylation of ACPA IgG to that of non-ACPA IgG from the same patients, and of healthy controls, in an IgG isoform-specific manner. We isolated ACPA and normal serum IgG, digested by trypsin, and separated the resulting peptide mixture by a reversed-phase nanoLC coupled to a Bruker Maxis II Q-TOF, and determined the relative abundance of glycoforms. The paired analysis of galactosylation and sialylation of the IgG subclasses of ACPA and non-ACPA IgG has shown a significant, moderate negative correlation with the inflammatory markers, the level of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), as well as with rheuma-factor (RF), but not with the disease activity score (DAS) or cyclic citrullinated peptide specific antibodies (anti-CCP). However, we detected a significant negative correlation between glycosylation and DAS in the non-ACPA IgG fractions. Furthermore, the isoform-specific analysis revealed additional insight into the changes of the glycosylation features of IgG in RA: changes in the frequencies of the bisecting GlcNAc unit between sample groups could be explained by only the IgG1 isoform; while invariance in fucosylation is the result of the superposition of two isoforms with opposite changes. These results highlight the importance of analyzing immunoglobulin glycosylation in an isoform-specific manner.

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure–function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

In the last half-century, capillary gel electrophoresis (CGE) became a versatile and high-performance analytical platform for the separation of complex biomolecular mixtures featuring rapid separations, high efficiency, and small sample consumption. Integrating a pore-size gradient mechanism in CGE makes it possible to achieve enhanced selectivity of polyionic macromolecules such as SDS-proteins and nucleic acids. This review provides a comprehensive overview of the theoretical foundations and operational principles of capillary pore-size gradient gel electrophoresis (CGGE), including the physicochemical basis of gradient formation, the influence of pore-size distributions on analyte mobility, and the challenges of generating stable, reproducible gradients in narrow-bore capillaries. Instrumental considerations such as capillary surface treatment, gradient filling and polymerization strategies, temperature and voltage control, detection modalities, and method-development frameworks are discussed in detail, emphasizing their critical impact on analytical performance and reproducibility. Key application areas in bioanalytical chemistry are highlighted, covering nucleic acid analysis and peptide/protein characterization. CGGE offers unique analytical advantages where fine molecular discrimination, tunable selectivity, and high resolution in a broad molecular weight range are required.