engleski

High-throughput LC-MS based analysis of plasma protein glycosylation

High-throughput glycomics gained a momentum in the last 15 years with the development of the methods adapted and optimized for the 96-well format (Ruhaak et al 2008 ; Royle et al 2008 ; Ruhaak et al 2010). Analytical processes were optimized on human plasma as major sample for the analysis primarily having glycans as new potential biomarkers in the focus of the research. Since then number of different methods were developed and optimized on different analytical platforms covering liquid chromatography with fluorescent detection, capillary electrophoresis with fluorescent detection or hyphenated to mass spectrometry, mass spectrometry based analytical approaches both with and without LC separation and lectin arrays (Trbojević-Akmačić et al 2022). In the last couple of years in our lab we introduced number of analytical techniques covering several plasma proteins, both human and rodent. Immunoglobulin G as the major analyte isolated from human, mouse and rat plasma, analysed on released glycan level and on the site-specific N- glycosylation level using UHPLC-FLR and LC-MS, respectively (Habazin et al 2021). Just recently, analytical approaches for two additional human plasma proteins, enriched to a higher level but not completely purified, were introduced for the analysis of glycopeptides. Methods for the Alpha- 1-acid glycoprotein (AGP) and Complement C3 site- specific N-glycosylation LC-MS analysis were proven on several large cohorts as qualitative analytical methods capable of detecting biologically meaningful data (Keser et al 2021 ; Šoić et al 2022). Glycosylation is one of the essential factors for the functioning of every organism and the development of effective analytical methods for qualitative and quantitative detection of glycans and their changes under various physiological and pathological conditions is a prerequisite for the successful research and introduction of glycans as clinically relevant biomarkers.

High-throughput ; LC-MS ; N-glycosylation ; glycoprotein

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