目前

O 糖 分析 L udger 采用 O 糖 释放 的 方法：

1

我们 用 的 是 LL ORELA A2 试剂盒， 而且 我们 实验 是 经过 反复 验证 的， 我们 收集 O 糖 后 如

何 计算 得率 也是 经常 验证 的。

The O

glycans that are released by Orela have non reduced termini. This means that they can be

fluorescently labelled and analysed by UHPLC. One way to calculate the yield would be to label

some Ludger BioQuant Chitotriose standard (BQ CHITOTRIOSE 01) alongside the released and

cleaned up O glycans and calculate the yield based on the quantified standard. Another way wou ld

be to run a Ludger BioQuant 2AB labelled Chitotriose standard or Ludger BioQuant 2AA labelled

Chitotriose standard alongside the 2AB or 2AA labelled O glycans and calculate the yield ( 请 见 产

品 使用 说明 http://www.ludger.com/docs/products/ll/ll orela a2/ll orela a2 guide v.2.1.pdf

2

而且 不 必 先 释放 N 糖， N 糖 存在 不会 影响 O 糖 释放。 此 试剂盒释放糖链 原理 是： The O

glycans are released from glycoproteins by ORela reagent by β elimination based procedure.

说明书

中 操作 的 在最后一步， acidification 之前糖胺（ glycosylamine derivatives ）是仅在糖链

与肽段断开的位置含有胺还是其他部位也有？ T he ORela reagent reacts at the link between the

glycan and peptide backbone to liberate glycans as the glycosylamine. The acid labile

glycosylamine derivative is hydrolysed to produce the free glycan.

3

O 糖最低检测浓度是多少？（样品量）

We recommend using at least 100 µg of glycoprotein sample (based on releases we have done

using bovine fetuin glycoprotein). Following release and CEX cleanup and fluorescent labelling

and SPE cleanup, the samples should be dried down and resuspended in 50 100 µL of water.

UHPLC chromatograms can then be obtained by injecting an amount of this concentrated

sample solution.

The amount of glycoprotein sample you can use will depend on the number of glycosylation

sites in the sample an d how glycosylated the sites will be. With a more heavily glycosylated

sample, less can be used. We would recommend the user to determine the lowest amount of

sample they can use for the Orela release as all glycoproteins are different.