Frommhagen Matthias, van Erven Gijs, Sanders Mark, van Berkel Willem J H, Kabel Mirjam A, Gruppen Harry
Laboratory of Food Chemistry, Wageningen University & Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.
Carbohydr Res. 2017 Aug 7;448:191-199. doi: 10.1016/j.carres.2017.03.006. Epub 2017 Mar 6.
Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides.
裂解多糖单加氧酶（LPMOs）能够通过氧化C1和/或C4原子来裂解难降解的多糖，如纤维素。对所得产物的分析需要多种分析技术。到目前为止，这些技术主要集中于非氧化和C1氧化低聚糖的鉴定。C4氧化葡糖低聚糖的分析大多通过高压阴离子交换色谱法（HPAEC）进行。然而，HPAEC分析过程中使用的碱性条件会导致C4氧化葡糖低聚糖的互变异构，这限制了该技术的应用。在此，我们描述了反相超高效液相色谱（RP-UHPLC）与非还原型2-氨基苯甲酰胺（2-AB）标记相结合的应用。非还原型2-AB标记能够将C4氧化葡糖低聚糖与其非氧化对应物分离。此外，RP-UHPLC不需要缓冲流动相，这降低了质谱（MS）的灵敏度。与其他技术如亲水相互作用液相色谱和与MS联用的多孔石墨化碳相比，这被视为一个优势。RP-UHPLC与紫外检测和质谱联用能够鉴定标记的非氧化和C4氧化低聚糖。非还原标记使LPMO氧化低聚糖C4位的酮保持完整，而选择性还原剂如三乙酰氧基硼氢化钠（STAB）则会还原该酮基。我们的结果表明，RP-UHPLC-UV-ESI-MS与非还原型2-AB标记相结合是一种适用于分离和鉴定LPMO产生的C4氧化葡糖低聚糖的技术。
