Selective Enrichment of Glycopeptides Using Aluminum Oxide
Selective Enrichment of Glycopeptides Using Aluminum Oxide
复制标题
使用氧化铝选择性富集糖肽
DOI：
10.6023/a12121103
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发表时间：
2013-03
影响因子：
2.5
通讯作者：
Liang Xinmiao
中科院分区：
文献类型：
--
作者：
Zhao Xu;Jiang Wuhui;Yu Long;Zou Lijuan;Li Xiuling;Liang Xinmiao
蛋白糖基化与细胞周期、免疫调节、细胞识别和细胞粘附等密切相关。异常糖基化表达与许多疾病有关，如类风湿关节炎、癌症和慢性阻塞性肺疾病。因此，蛋白质糖基化的表征对了解生命过程非常重要。然而，由于复杂样品中糖肽类化合物的浓度较低，且非糖肽类化合物的信号被抑制，使得质谱法对糖肽类化合物的检测存在困难。因此，探索一种有效的糖肽富集技术是十分必要的。本研究采用氧化铝基材料选择性富集糖肽。首先，我们通过改变乙腈（ACN）的浓度来研究其保留机理。随着ACN浓度的降低，多肽被根据其亲水性被洗脱。此外，大部分非糖肽比高浓度ACN中的糖肽更早被洗脱，而在低浓度ACN中发现了糖肽。这一结果证明亲水性相互作用是氧化铝保留多肽的机制之一。其次，通过改变氢氧化铵的浓度，研究了氢氧化铵的保留机理。将ACN固定在高浓度，然后用不同浓度的氢氧化铵溶液洗脱肽段。在低浓度的氢氧化铵溶液中，没有发现肽。但在高浓度的氢氧化铵溶液中，糖肽和非糖肽同时被洗脱。这证明配体交换也是保留机制之一，在这种机制下，糖肽和非糖肽的保留没有区别。在此基础上，对固相萃取模型下的富集条件进行了优化。高浓度ACN与一定浓度的氢氧化铵溶液混合作为加载缓冲液，低浓度ACN与一定浓度的氢氧化铵溶液混合作为洗脱缓冲液。将氧化铝富集的糖肽数量与浓缩前的甘氨酸-肽数量和Sepharose富集的糖肽数量进行比较。在胰酶消化中，富集前共发现7个糖肽，氧化铝共发现16个糖肽，Sepharose共发现14个糖肽。在胰蛋白酶IgG消化中，富集前发现2个糖肽，氧化铝发现12个糖肽，Sepharose发现4个糖肽。综上所述，氧化铝基富集糖肽的方法具有选择性高、覆盖范围广的特点。关键词氧化铝;糖肤;丰富;质谱;肽
Protein glycosylation is significantly associated with cells cycle, immune regulation, cells recognition and cells adhesion. Aberrant glycosylation expression is involved in many diseases, such as rheumatoid arthritis, cancer, and chronic obstructive pulmonary disease. Thus, characterization of the protein glycosylation is very important to understand the life process. However, detection of glycopeptides is difficult by mass spectrometry because of the low concentration in complex sample and the suppressed signal by non-glycopeptides. Therefore, it is essential to explore an effective technique for glyco- peptide enrichment. In this study, an alumina based-materials was used to selectively enrich glycopeptides. Firstly, we inves- tigated the retention mechanism by changing the concentration of acetonitrile (ACN). With the decreased concentration of ACN, it was found that peptides were eluted according to their hydrophilicity. Moreover, most of the non-glycopeptides were eluted earlier than the glycopeptides in the high concentration of ACN fraction and the glycopeptides were found in the low concentration of ACN fraction. This result proved that hydrophilic interaction is one of the retention mechanisms for peptides retained by alumina. Secondly, we investigated the retention mechanism by changing the concentration of ammonium hy- droxide. ACN were fixed at a high concentration and peptides were subsequently eluted with different concentration of am- monium hydroxide solution. At a low concentration of ammonium hydroxide solution, the peptides were no found. But at a high concentration of ammonium hydroxide solution, the glycopeptides and non-glycopeptides were eluted simultaneously. This proved that the ligand exchange is also one of the retention mechanisms, where the retention of the glycopeptides and non-glycopeptides showed no different under this mechanism. Based on the above mentioned results, the enrichment condi- tion was optimized under the model of solid-phase extraction. High concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as loading buffer and low concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as elution buffer. The number of glycopeptides enriched by alumina were compared with gly- copeptides before enrichment and enriched by Sepharose. In the tryptic HRP digest, 7 glycopeptides were found before en- richment, 16 glycopeptides were found by alumina and 14 glycopeptides were found by Sepharose. In the tryptic IgG digest, 2 glycopeptides were found before enrichment, 12 glycopeptides were found by alumina and 4 glycopeptides were found by Sepharose. In conclusion, alumina based-method used to enrich glycopeptides has high selection and wide coverage. Keywords alumina; glycopeptides; enrichment; mass spectrometry; peptides