Bank Stephanie, Heller Eberhard, Memmel Elisabeth, Seibel Jürgen, Holzgrabe Ulrike, Kapková Petra
Institute of Pharmacy and Food Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074, Würzburg, Germany.
Rapid Commun Mass Spectrom. 2014 Aug 15;28(15):1745-56. doi: 10.1002/rcm.6955.
Successful structural characterization of glycans often requires derivatization prior to mass spectrometric analysis. Here we report on a new derivatization reagent for glycans, biotinylated isonicotinic hydrazide, allowing glycan analysis by both mass spectrometry (MS) and biochemically. Fragmentation behavior in MS and its use in structural elucidation were investigated and compared with other labels.
Glycans, released from ribonuclease B and ovalbumin, were derivatized with hydrazine labels (isoniazid (INH), biotinylated isonicotinic hydrazide (BINH) and biotinamidocaproylhydrazide (BACH)). In addition, native counterparts and 2-aminobenzamide (2-AB) derivatives were prepared. Comparative matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF) experiments were carried out to investigate the fragmentation pattern of the derivatives. Finally, the capability of BINH derivatives to bind lectins was explored.
Generally, derivatization provided beneficial enhancement in the mass spectrometric signal intensity as compared to native counterparts. The mass spectrometric fragmentation varied with the kind of label used. The most significant structure-revealing ions (cross-ring cleavages) were observed in the spectra of BINH derivatives, whereas mainly glycosidic cleavages were found with native form of glycans and 2-AB derivatives.
Hydrazine derivatization provided the means to obtain structurally informative fragment ions. Due to BINH derivatization, specific fragments of the isomers allowed the identification of diverse glycans. The derivatization reaction can be carried out without the need for purification. The biotin residue of BINH enabled for biochemical studies, i.e. protein-glycan interactions.
聚糖的成功结构表征通常需要在质谱分析之前进行衍生化。在此，我们报道一种新的聚糖衍生化试剂——生物素化异烟肼，它可通过质谱（MS）和生化方法进行聚糖分析。研究了其在质谱中的裂解行为及其在结构解析中的应用，并与其他标记物进行了比较。
从核糖核酸酶B和卵清蛋白中释放的聚糖用肼类标记物（异烟肼（INH）、生物素化异烟肼（BINH）和生物素氨基己酰肼（BACH））进行衍生化。此外，还制备了天然聚糖对应物和2-氨基苯甲酰胺（2-AB）衍生物。进行了比较基质辅助激光解吸/电离串联飞行时间（MALDI TOF/TOF）实验，以研究衍生物的裂解模式。最后，探索了BINH衍生物与凝集素结合的能力。
一般来说，与天然聚糖对应物相比，衍生化在质谱信号强度方面提供了有益的增强。质谱裂解随所用标记物的种类而变化。在BINH衍生物的光谱中观察到最显著的揭示结构的离子（交叉环裂解），而在聚糖的天然形式和2-AB衍生物的光谱中主要发现糖苷键裂解。
肼衍生化提供了获得具有结构信息的碎片离子的方法。由于BINH衍生化，异构体的特定碎片允许鉴定多种聚糖。衍生化反应无需纯化即可进行。BINH的生物素残基可用于生化研究，即蛋白质-聚糖相互作用。
