Quéméner Bernard, Vigouroux Jacqueline, Rathahao Estelle, Tabet Jean Claude, Dimitrijevic Aleksandra, Lahaye Marc
INRA, Biopolymères, Interactions, Assemblage, Rue de la Géraudière BP 71627, F-44316, Nantes, France.
J Mass Spectrom. 2015 Jan;50(1):247-64. doi: 10.1002/jms.3528.
Xyloglucans of apple, tomato, bilberry and tamarind were hydrolyzed by commercial endo β-1-4-D-endoglucanase. The xylo-gluco-oligosaccharides (XylGos) released were separated on CarboPac PA 200 column in less than 15 min, and, after purification, they were structurally characterized by negative electrospray ionization mass spectrometry using a quadrupole time-of-flight (ESI-Q-TOF), a hybrid linear ion trap (LTQ)/Orbitrap and a hybrid quadrupole Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. In order to corroborate the fragmentation routes observed on XylGos, some commercial galacto-manno-oligosaccharides (GalMOs) and glucurono-xylo-oligosaccharides were also studied. The fragmentation pathways of the ionized GalMos were similar to those of XylGos ones. The product ion spectra were mainly characterized by prominent double cleavage (D) ions corresponding to the entire inner side chains. The directed fragmentation from the reducing end to the other end was observed for the main glycosylated backbone but also for the side-chains, allowing their complete sequencing. Relevant cross-ring cleavage ions from (0,2)X(j)-type revealed to be diagnostic of the 1-2-linked- glycosyl units from XylGos together with the 1-2-linked glucuronic acid unit from glucuronoxylans. Resonant activation in the LTQ Orbitrap allowed not only determining the type of all linkages but also the O-acetyl group location on fucosylated side-chains. Moreover, the fragmentation of the different side chains using the MS(n) capabilities of the LTQ/Orbitrap analyzer also allowed differentiating terminal arabinosyl and xylosyl substituents inside S and U side-chains of XylGos, respectively. The CID spectra obtained were very informative for distinction of isomeric structures differing only in their substitution pattern. These features together makes the fragmentation in negative ionization mode a relevant and powerful technique useful to highlight the subtle structural changes generally observed during the development of plant organs such as during fruit ripening and for the screening of cell wall mutants with altered hemicellulose structure.
苹果、番茄、越橘和罗望子中的木葡聚糖被商业内切β-1,4-D-内切葡聚糖酶水解。释放出的木葡寡糖（XylGos）在CarboPac PA 200柱上于不到15分钟内分离，纯化后，使用四极杆飞行时间（ESI-Q-TOF）、混合线性离子阱（LTQ）/轨道阱和混合四极杆傅里叶变换离子回旋共振（FT-ICR）质谱仪通过负电喷雾电离质谱对其进行结构表征。为了证实XylGos上观察到的裂解途径，还研究了一些商业半乳甘露寡糖（GalMOs）和葡糖醛酸木寡糖。离子化GalMos的裂解途径与XylGos的相似。产物离子光谱的主要特征是对应于整个内侧链的突出双裂解（D）离子。观察到从还原端到另一端的定向裂解，不仅针对主要的糖基化主链，也针对侧链，从而实现其完全测序。来自(0,2)X(j)型的相关跨环裂解离子被证明是XylGos中1-2连接糖基单元以及葡糖醛酸木聚糖中1-2连接葡糖醛酸单元的诊断特征。LTQ轨道阱中的共振激活不仅能够确定所有连接的类型，还能确定岩藻糖基化侧链上O-乙酰基的位置。此外，利用LTQ/轨道阱分析仪的MS(n)功能对不同侧链进行裂解，还能分别区分XylGos的S和U侧链内的末端阿拉伯糖基和木糖基取代基。获得的CID光谱对于区分仅在取代模式上不同的异构体结构非常有帮助。这些特征共同使得负电离模式下的裂解成为一种相关且强大的技术，有助于突出植物器官发育过程中（如果实成熟期间）普遍观察到的细微结构变化，并用于筛选具有改变的半纤维素结构的细胞壁突变体。
