Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study

Abstract

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a five-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2 percent. The highest CVs ranging from 16 to 29 percent accompanied low abundance glycan peaks with the individual relative peak area below 1 percent that together made up for less than 2 percent of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Immunoglobulin G glycome composition in transition from premenopause to postmenopause

“…Deglycosylation, RapiFluor-MS labelling and purification of IgG N-glycans were performed using the GlycoWorks RapiFluor-MS N-Glycan Kit obtained from Waters Corporation (USA). The entire procedure was done following Waters’ protocol (Waters Corporation, 2017, https://www.waters.com/webassets/cms/ support/docs/715004793en.pdf), which was further adapted to suit the high-throughput analysis in the 96-well PCR plate format ( Deriš et al., 2021 ). Isolated and dried IgG was first resuspended in 10.8 μL ultrapure water, swiftly denatured with 3 μL 5 % RapiGest SF solution in a 3 min reaction at 99°C and enzymatically deglycosylated with 1.2 μL GlycoWorks Rapid PNGase F in a 5 min reaction at 50°C.…”

IgG N-glycome changes during the course of severe COVID-19: An observational study

“…The protocol for this analysis was described in detail by Deriš and colleagues. 21 Briefly, dried IgG eluate (average mass of 15 µg) was resuspended in ultrapure water and 5 % GlycoWorks RapiGest SF solution (Waters, USA)was added to each sample to denature IgG. N-glycans from samples were enzymatically released from IgG by GlycoWorks Rapid PNGase F (Waters, USA) and labelled with GlycoWorks RapiFluor-MS Solution (Waters, USA).…”

Differences in Immunoglobulin G Glycosylation Between Influenza and COVID-19 Patients

“…For COVID-19 samples, glycan release, and labeling, a GlycoWorks RapiFluor-MS N -Glycan Kit (Waters, USA) was used. The protocol for this analysis has been described in detail by Deriš et al [25] . In brief, dried IgG eluate (with an average mass of 15 µg) was resuspended in ultrapure water and 5% RapiGest SF solution (Waters).…”