Journal of Proteomics ( IF 2.8 ) Pub Date : 2021-01-06 , DOI: 10.1016/j.jprot.2020.104098 Dongqi Liu 1 , Gang Liu 2 , Yuqing Li 2 , Yue Wang 2 , Yuanyuan Zheng 2 , Shanshan Sha 2 , Wenzhe Li 2 , Akihiko Kameyama 3 , Weijie Dong 2
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Previously, we developed a novel separation technique, namely, supported molecular matrix electrophoresis (SMME), which separates mucins on a PVDF membrane that impregnated with a hydrophilic polymer (such as polyvinyl alcohol), so it has the characteristics that are compatible with glycan analysis of the separated bands. Here, we describe the first instance of the application of SMME to mouse sera fractionation and demonstrate their differences from the pooled human sera fractionation by SMME. Furthermore, we have developed a fixation method for the lectin blotting of SMME-separated glycoproteins by immersing the SMME membranes into acetone solvent followed by heating. It showed that the amount of protein samples required for SMME were reduced more than 4-fold than that of the process of SDS-PAGE. We applied these techniques for the detection of glycosylation patterns of serum proteins from Fut8+/+ and Fut8−/− mice, further analyzed N-linked and O-linked glycans from the separated γ-bands by mass spectrometry, and demonstrated that there are α2,8-sialylated O-glycans contained in mouse sera glycoproteins. SMME can provide simple, rapid sera fractionation, glycan profiling differences between the bands of two samples and a new insight into the underlying mechanism that responsible for related diseases.
Significance
We describe that the first application of SMME can separate mouse serum proteins into six bands and identify the major protein components of each fraction in mouse serum separated by SMME. Furthermore, we successfully developed a fixation method for lectin blotting of SMME-separated glycoproteins and applied to the detection of glycosylation patterns of serum glycoproteins from Fut8+/+ and Fut8−/− mice, also, the method is promising for detecting glycan profiling differences between two samples in both research and clinical settings.
中文翻译：
支持的分子基质电泳分离小鼠血清糖蛋白的快速糖基化分析
以前，我们开发了一种新的分离技术，即支持分子基质电泳（SMME），该技术可将PVDF膜上的粘蛋白分离，该膜上浸有亲水性聚合物（例如聚乙烯醇），因此它具有与聚糖分析兼容的特性。分离的乐队。在这里，我们描述了将SMME应用于小鼠血清分级分离的第一个实例，并证明了它们与SMME合并的人类血清分级分离的差异。此外，我们已经开发了一种将SMME分离的糖蛋白凝集素印迹的固定方法，方法是将SMME膜浸入丙酮溶剂中，然后加热。结果表明，SMME所需的蛋白质样品量比SDS-PAGE过程减少了4倍以上。Fut8 + / +和Fut8 -/-小鼠，通过质谱进一步分析了分离的γ带中的N-连接和O-连接的聚糖，并证明小鼠血清糖蛋白中含有α2,8-唾液酸化的O-聚糖。SMME可以提供简单，快速的血清分级分离，两个样品条带之间的聚糖谱分析差异，以及对引起相关疾病的潜在机制的新见解。
意义
我们描述了SMME的首次应用可以将小鼠血清蛋白分为6个带，并鉴定出被SMME分离的小鼠血清中每个组分的主要蛋白成分。此外，我们成功开发出了SMME分离的糖蛋白的凝集素印迹固定方法，并用于检测Fut8 + / +和Fut8 -/-小鼠血清糖蛋白的糖基化模式，该方法有望用于检测聚糖谱分析差异在研究和临床环境中的两个样本之间。
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