Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization.
Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization.
复制标题
用于并行蛋白质组和 N-糖组表征的通用固相可逆样品制备。
DOI：
10.1021/acs.jproteome.5b00865
复制
发表时间：
2016
影响因子：
4.4
通讯作者：
Lee,RichardS
中科院分区：
文献类型：
--
作者：
Zhou,Hui;Morley,Samantha;Kostel,Stephen;Freeman,MichaelR;Joshi,Vivek;Brewster,David;Lee,RichardS
我们描述了一种新型的固相可逆样品制备（SRS）平台，该平台可以快速制备几乎所有蛋白质样品的同时蛋白质组和n -糖苷表征。SRS利用一种独特的功能化硅基头，对蛋白质有很强的亲和力，对肽和其他小分子几乎没有亲和力。通过利用蛋白质和肽之间固有的大小差异，SRS允许高容量的蛋白质结合，快速去除小分子（洗涤剂，代谢物，盐，肽等），广泛的操作，包括对珠状结合蛋白质的酶和化学处理，以及n -聚糖和肽的容易回收。SRS在广泛的样品中进行了评估，包括糖蛋白、细胞裂解液、小鼠组织和人类尿液。SRS还与定量策略相结合，以研究DU145前列腺癌细胞与其膜片沉默的对应物之间的差异。先前的研究表明，在DU145中，膜片3沉默诱导转变为与肿瘤进展和转移相关的变形虫表型。在这项初步研究中，我们确定了它们之间不同的蛋白质组学和n -糖组学改变。转移相关酪氨酸激酶受体ephrin- A型受体（EPHA2）在DIAPH3沉默的细胞中高度上调，表明EPHA2与DIAPH3之间可能存在联系。此外，鉴定出n -糖苷的明显改变，表明在DIAPH3和糖基转移酶网络之间存在交联。
We describe a novel solid-phase reversible sample-prep (SRS) platform that enables rapid sample preparation for concurrent proteome and N-glycome characterization for nearly all protein samples. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal to no affinity for peptides and other small molecules. By leveraging this inherent size difference between proteins and peptides, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, peptides, etc.), extensive manipulation including enzymatic and chemical treatments on bead-bound proteins, and easy recovery of N-glycans and peptides. SRS was evaluated in a wide range of samples including glycoproteins, cell lysate, murine tissues, and human urine. SRS was also coupled to a quantitative strategy to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Previous studies suggested that DIAPH3 silencing in DU145 induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this pilot study we identified distinct proteomic and N-glycomic alterations between them. A metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated in DIAPH3-silenced cells, indicating a possible connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting cross-links between DIAPH3 and glycosyltransferase networks.