N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

N-聚糖分析可用于揭示疾病相关的生物标志物，以及用于分析支持诊断称为先天性糖基化障碍 (CDG) 的聚糖生物合成遗传疾病的独特变化。基于优先耦合到电喷雾电离 (ESI)-质谱 (MS) 的液相色谱 (LC) 策略已成为N-聚糖鉴定和表征的强大分析方法。为了提高检测灵敏度，糖链通常在 LC-MS 分析之前用功能标签进行标记。由于大多数衍生化技术非常耗时，因此开发了一些商业分析试剂盒来加速N-去糖基化和N-具有药物和生物学意义的糖蛋白的聚糖标记，例如单克隆抗体 (mAb)。我们利用Rapi Fluor-MS (RFMS) 的分析能力，通过稍微修改的方案，对特定 CDG 患者 (MAN1B1-CDG、ALG12-CDG、MOGS) 的总血清和单一血清糖蛋白的N-聚糖进行详细表征-CDG、TMEM199-CDG)。该策略通过亲水相互作用色谱 (HILIC)-UPLC-ESI-MS 分离 RFMS 衍生的N-聚糖，使我们能够揭示患者血清释放的 N-聚糖的结构细节。聚糖，从而扩展了当前关于这些个体糖基化疾病中聚糖谱的知识。在某些情况下，所应用的方法能够区分结构异构体和连接类型不同的异构体。此处报告的所有应用表明，RFMS 方法与 HILIC-UPLC-ESI-MS 相结合，代表了一种灵敏的高通量血清N-糖组分析方法，也是聚糖检测和分离的一种有价值的选择，尤其是异构物质。