Zhang Yue, Li Jiyong, Yu Yuanhang, Xie Rong, Liao Han, Zhang Bo, Chen Jianying
Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430022 China
Department of Breast and Thyroid Surgery, Huangpi People's Hospital, Jianghan University Wuhan 430300 China.
RSC Adv. 2020 Jun 9;10(37):22176-22182. doi: 10.1039/d0ra01048k. eCollection 2020 Jun 8.
Simultaneous profiling of protein phosphorylation and glycosylation is very important to elucidate the bio-functions of these proteins. However, simultaneous enrichment of glyco- and phosphopeptides is the bottleneck in proteomics because of the low abundance of these species and ion suppression from non-modified peptides in mass spectrometry (MS). In this study, Fe immobilized hydrophilic interaction chromatography (HILIC) materials (termed polySD-SiO, recently reported in our lab) and polySD-SiO in the HILIC mode were employed for the simultaneous enrichment and subsequent separation of glyco- and phosphopeptides. The Fe immobilized polySD-SiO could selectively enrich glycopeptides and phosphopeptides and the co-enriched peptides were further fractionated with polySD-SiO in the HILIC mode. With the established method, glyco- and phosphopeptides were well enriched and divided into two fractions even from tryptic digests of a-casein, fetuin and BSA at a molar ratio of 1 : 2 : 400. Application of the established method to HeLa cell lysate resulted in a total of 1903 phosphopeptides and 141 glycosylation sites. These results demonstrate that the established method could selectively and simultaneously enrich and fractionate glyco- and phosphopeptides from complex peptide mixtures.
同时分析蛋白质磷酸化和糖基化对于阐明这些蛋白质的生物学功能非常重要。然而，由于这些物质丰度低以及质谱（MS）中未修饰肽的离子抑制作用，糖肽和磷酸肽的同时富集是蛋白质组学中的瓶颈。在本研究中，采用铁固定化亲水相互作用色谱（HILIC）材料（即我们实验室最近报道的聚SD - SiO）以及处于HILIC模式的聚SD - SiO对糖肽和磷酸肽进行同时富集及后续分离。铁固定化聚SD - SiO能够选择性地富集糖肽和磷酸肽，并且将共富集的肽在HILIC模式下用聚SD - SiO进一步分级分离。利用所建立的方法，即使从α - 酪蛋白、胎球蛋白和牛血清白蛋白按1∶2∶400摩尔比的胰蛋白酶消化物中，糖肽和磷酸肽也能得到良好的富集并分成两个级分。将所建立的方法应用于HeLa细胞裂解物，共得到1903个磷酸肽和141个糖基化位点。这些结果表明，所建立的方法能够从复杂的肽混合物中选择性地同时富集和分级分离糖肽和磷酸肽。
