engleski

Comparison of 2-Aminobenzamide, Procainamide and RapiFluor-MS as Derivatizing Agents for High-Throughput HILIC-UPLC-FLR-MS N-glycan Analysis

Rising awareness of universal importance of protein N-glycosylation governs the development and further advances in N-glycan analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds – 2-aminiobenzamide (2-AB) and procainamide (ProA), and recently introduced RapiFluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derived, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification, repeatability and labeling efficiency for all three labels. We also tested the performance of all three labels in a high-throughput setting on 68 different IgG samples, all in duplicates, and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS, respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limit of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. Furthermore, the results indicated that labeling efficiency was very similar for all three labels. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.

N-glycans ; 2-aminobenzamide ; procainamide ; RapiFluor-MS ; IgG ; HILIC ; fluorescence ; mass spectrometry

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