Naslov
Comparison of 2-aminobenzamide, procainamide and Rapifluor-MS as fluorescent labels for high-throughput HILIC-UPLC N-glycan analysis

Autori
Pavić, Tamara ; Keser, Toma ; Gornik, Olga ; Lauc, Gordan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
12th Jenner Glycobiology and Medicine Symposium “Translational Glycobiology – From Bench to Bedside” Book of Abstracts / - , 2017, 100-100

Skup
12th Jenner Glycobiology and Medicine Symposium “Translational Glycobiology – From Bench to Bedside”

Mjesto i datum
Zagreb, Hrvatska; Dubrovnik, Hrvatska, 06.05.2017. - 09.05.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
2-aminobenzamide ; procainamide ; Rapifluor-MS ; N-glycans ; N-glycome ; Immunoglobulin G ; high-throughput

Sažetak
Rising awareness of universal importance of protein N-glycosylation governs the development and further advances in N-glycan analysis. Nowadays it’s well known that correct glycosylation is essential for proper protein function, which emanates with its important role in many physiological processes and pathophysiology of multiple common complex diseases. In the vast majority of cases, the N- glycosylation profiles are analysed from enzymatically released glycans, which can be further derivatized, in order to enhance the sensitivity of the analysis. Techniques wherein fluorescently derivatized N-glycans are profiled using hydrophilic interaction chromatography with fluorescence detection (HILIC-UPLC-FLR) are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the sensitivity and performance of frequently used labelling compounds – 2- aminiobenzamide (2-AB) and procainamide (ProA), and recently introduced RapiFluor-MS fluorescent tag, in a high-throughput setting. For that purpose, we employed commercial IgG standard and inhouse prepared IgG, as a model glycoprotein. All samples were analysed in triplicates using different amounts of starting material (0.1-500 μg of IgG for 2-AB and ProA labelling ; 0.1-30 μg of IgG for RapiFluor-MS labelling). In all three parallel experiments N-glycans were released by PNGase F, fluorescently derived, purified by HILIC-SPE and profiled using HILIC-UPLC-FLR. In general, ProA derived glycans showed 15-fold and 4-fold higher signal intensities compared to 2-AB and RapiFluor-MS, respectively. All labelling procedures exhibited good and comparable reproducibility. Reliable and reproducible quantification of individual glycans is possible when profiling glycans released from 1 μg of IgG in the case of 2-AB labelling, 0.2 μg for ProA and 0.3 μg for RapiFluor-MS labelling. Although ProA showed enhanced signal intensity, RapiFluor-MS has comparable limit of quantification, whilst 2-AB exhibited the lowest sensitivity.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti, Farmacija

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