Yu Zixiang, Zhao Xinyuan, Tian Fang, Zhao Yang, Zhang Yong, Huang Yi, Qian Xiaohong, Ying Wantao
Anhui Medical University, No. 81 Meishan Road, Hefei, Anhui, 230032, China.
National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
Anal Bioanal Chem. 2017 May;409(12):3077-3087. doi: 10.1007/s00216-017-0195-z. Epub 2017 Mar 3.
Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC-MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.
糖蛋白结构的详细表征需要确定糖基化位点以及与每个位点相关的聚糖结构。在这项工作中，我们开发了一种用于表征复杂蛋白质组样品中完整N - 糖肽的分析策略。第一步，使用ZIC - HILIC富集胰蛋白酶水解糖肽。其次，一部分糖肽用内切糖苷酶H（Endo H）处理以去除高甘露糖（Man）和杂合N - 连接聚糖。第三步，将一部分经Endo H处理的糖肽在O水中进一步进行PNGase F处理，以去除剩余的复杂聚糖。完整糖肽和去糖基化肽通过纳米反相液相色谱 - 串联质谱（nano - RPLC - MS/MS）进行分析，聚糖结构和肽序列通过使用Byonic或pFind工具进行鉴定。内切糖苷酶的顺序消化提供了候选糖基化位点信息以及每个糖肽上糖型的指示，从而有助于限制数据库搜索空间并提高完整糖肽鉴定的可信度。我们使用核糖核酸酶B（RNase B）和免疫球蛋白G（IgG）证明了该方法的有效性，并将这种顺序消化策略应用于从肝癌细胞系（HepG2）中鉴定糖肽。我们从HepG2细胞中鉴定出4514个来自947个糖基化位点的完整糖肽和1011个独特的肽序列。获得特定糖基化位点处不同糖型的强度以达到位点特异性糖型的占有率。这些结果表明我们的方法可用于表征复杂样品中位点特异性蛋白质糖基化。图形摘要通过整合完整糖肽、碎片离子筛选和内切糖苷酶消化的信息，可以显著提高鉴定的可靠性。我们同时通过每个糖肽的质谱响应信号量化位点特异性糖型占有率。
