Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides
Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides
复制标题
DOI：
10.1016/j.carres.2005.10.011
复制
发表时间：
2006-01-16
影响因子：
3.1
通讯作者：
Thomas, ORT
中科院分区：
文献类型：
--
作者：
Guillaumie, F;Justesen, SFL;Thomas, ORT
这项工作介绍了果胶低聚体的优化分离，它们的基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)分析，它们随后固定在载体上，以及我们迈向固体载体辅助测序的第一步。采用常压强阴离子交换树脂Source15Q与甲酸铵缓冲液(AF)联用，对果胶酶解产物中的不饱和和饱和果胶低聚半乳糖苷(OGAS)进行了分离。通常，DPS从5到19的多毫克量的限定尺寸的Ogas以极高的纯度(>；95%)生产。用MALDI-TOF MS对OGAS进行洗脱，用MALDI-TOF MS直接对峰进行分析，将纯化的OGAS(DP5-7)化学选择性地固定在端氨氧基的聚乙二醇聚丙烯酰胺(PEGA)载体上。固相锚定发生在低聚糖的还原末端，并导致了肟键的形成。很高的偶联产率证实了氨氧基-PEGA树脂对不同长度的Ogas的固定化具有普遍的适用性。OGA功能化的PEGA载体随后在40或60℃下用AQ TFA处理，并用ESIMS分析从载体中释放的化学降解产物。在所有情况下，原始的OGA都被降解成不同大小的更小的齐聚物，直到单体。这项工作说明了最终以固体支持辅助低聚糖测序为目标的战略的一些基本原则。(C)2005爱思唯尔有限公司。保留所有权利。
This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (> 95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides. (c) 2005 Elsevier Ltd. All rights reserved.