Buck-Wiese Hagen, Fanuel Mathieu, Liebeke Manuel, Le Mai Hoang Kim, Pardo-Vargas Alonso, Seeberger Peter H, Hehemann Jan-Hendrik, Rogniaux Hélène, Jackson Glen P, Ropartz David
Max-Planck-Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany.
Marine Glycobiology, Marum Center for Marine Environmental Sciences, Leobener Strasse 8, 28359 Bremen, Germany.
J Am Soc Mass Spectrom. 2020 Jun 3;31(6):1249-1259. doi: 10.1021/jasms.0c00087. Epub 2020 May 4.
The connection between monosaccharides influences the structure, solubility, and biological function of carbohydrates. Although tandem mass spectrometry (MS/MS) often enables the compositional identification of carbohydrates, traditional MS/MS fragmentation methods fail to generate abundant cross-ring fragments of intrachain monosaccharides that could reveal carbohydrate connectivity. We examined the potential of helium-charge transfer dissociation (He-CTD) as a method of MS/MS to decipher the connectivity of β-1,4- and β-1,3-linked oligosaccharides. In contrast to collision-induced dissociation (CID), He-CTD of isolated oligosaccharide precursors produced both glycosidic and cross-ring cleavages of each monosaccharide. The radical-driven dissociation in He-CTD induced single cleavage events, without consecutive fragmentations, which facilitated structural interpretation. He-CTD of various standards up to a degree of polymerization of 7 showed that β-1,4- and β-1,3-linked carbohydrates can be distinguished based on diagnostic A fragment ions that are characteristic for β-1,4-linkages. Overall, fragment ion spectra from He-CTD contained sufficient information to infer the connectivity specifically for each glycosidic bond. When testing He-CTD to resolve the order of β-1,4- and β-1,3-linkages in mixed-linked oligosaccharide standards, He-CTD spectra sometimes provided less confident assignment of connectivity. Ion mobility spectrometry-mass spectrometry (IMS-MS) of the standards indicated that ambiguity in the He-CTD spectra was caused by isobaric impurities in the mixed-linked oligosaccharides. Radical-driven dissociation induced by He-CTD can thus expand MS/MS to carbohydrate linkage analysis, as demonstrated by the comprehensive fragment ion spectra on native oligosaccharides. The determination of connectivity in true unknowns would benefit from the separation of isobaric precursors, through UPLC or IMS, before linkage determination via He-CTD.
单糖之间的连接方式会影响碳水化合物的结构、溶解性和生物学功能。尽管串联质谱（MS/MS）通常能够实现碳水化合物的成分鉴定，但传统的MS/MS碎片化方法无法产生丰富的链内单糖交叉环碎片，而这些碎片可以揭示碳水化合物的连接性。我们研究了氦电荷转移解离（He-CTD）作为一种MS/MS方法来解析β-1,4-和β-1,3-连接的寡糖连接性的潜力。与碰撞诱导解离（CID）不同，分离的寡糖前体的He-CTD产生了每个单糖的糖苷键和交叉环裂解。He-CTD中自由基驱动的解离诱导了单次裂解事件，没有连续碎片化，这有助于结构解析。对聚合度高达7的各种标准品进行He-CTD分析表明，基于β-1,4-连接特有的诊断性A碎片离子，可以区分β-1,4-和β-1,3-连接的碳水化合物。总体而言，He-CTD的碎片离子光谱包含足够的信息来推断每个糖苷键的具体连接性。在测试He-CTD以解析混合连接寡糖标准品中β-1,4-和β-1,3-连接的顺序时，He-CTD光谱有时提供的连接性归属不太确定。对标准品的离子淌度质谱（IMS-MS）分析表明，He-CTD光谱中的模糊性是由混合连接寡糖中的等压杂质引起的。因此，He-CTD诱导的自由基驱动解离可以将MS/MS扩展到碳水化合物连接分析，天然寡糖的综合碎片离子光谱证明了这一点。对于真正未知物连接性的确定，在通过He-CTD进行连接测定之前，通过超高效液相色谱（UPLC）或IMS对等压前体进行分离将有助于分析。
