Two-step protease digestion and glycopeptide capture approach for accurate glycosite identification and glycoprotein sequence coverage improvement
Two-step protease digestion and glycopeptide capture approach for accurate glycosite identification and glycoprotein sequence coverage improvement
复制标题
两步蛋白酶消化和糖肽捕获方法可准确识别糖位点并提高糖蛋白序列覆盖度
DOI：
10.1016/j.talanta.2011.03.029
复制
发表时间：
2011-07-15
期刊：
影响因子：
6.1
通讯作者：
Yang, Pengyuan
中科院分区：
文献类型：
--
作者：
Chen, Yaohan;Cao, Jing;Yang, Pengyuan
开发了一种新的两步蛋白酶消化和糖肽捕获方法。它不同于传统的糖蛋白组学的胰酶消化、糖肽富集和鉴定方法。在这里，蛋白质首先被赖氨酸- c消化成相对较大的肽。其中的糖肽通过氧化聚糖被肼树脂选择性捕获。经过彻底的洗涤步骤后，胰蛋白酶作为第二蛋白酶从糖肽中原位释放非糖基化部分（称为lt -肽）。随后，树脂上剩余的部分糖肽被肽- n -糖苷酶f去糖基化并收集为dg -肽。最后，用质谱仪对LT-和dg -肽进行分析，实现糖蛋白和糖位点的鉴定。该方法应用于模型糖蛋白阳性验证后的细胞裂解液：从DG-和lt -馏分中鉴定出143个n -糖蛋白。在这些糖蛋白中，189个dg -peptide显示的n -糖位点得到了相邻lt -肽的进一步确认，同时使109个糖蛋白的序列覆盖率提高了350%（平均79.4%）。该方法通过对非糖肽（本文新引入的lt肽）和传统去糖肽的可控释放、分离鉴定和联合解释，不仅可以实现常规的n-糖苷位点鉴定，还可以进一步提供n-糖苷位点的证据，增加糖蛋白序列覆盖率。(C) 2011 Elsevier B.V.版权所有
A novel two-step protease digestion and glycopeptide capture approach has been developed. It is different from traditional tryptic digestion, glycopeptide enriching and identification approach in glycoproteomics. Here, proteins were first digested by Lys-C into relatively large peptides. Glycopeptides among them were selectively captured by hydrazide resin through oxidized glycans. After thorough washing steps, trypsin was used as a second protease to in situ release non-glycosylated part (named as LT-peptides) from glycopeptides. Subsequently, the remaining part of glycopeptides on resin was de-glycosylated by peptide-N-glycosidase F. and collected as DG-peptides. Finally, both LT- and DG-peptides could be analyzed by mass spectrometer, achieving glycoprotein and glycosite identification. The approach was applied to cell lysate after positive validation by a model glycoprotein: 143 N-glycoproteins identified from DG- and LT-fraction both. In those glycoproteins, 189 DG-peptide-revealed N-glycosites got further confirmation by neighboring LT-peptides, which, in the meantime, made 109 glycoproteins get improved sequence coverage with increase even up to 350% (averagely 79.4%). Through controllable release, separate identification and combined interpretation of non-glycopeptides (newly introduced LT-peptides here) and traditional de-glycopeptides, the approach could not only achieve routine N-glycosite identification, but also provide further proofs of N-glycosites and increase glycoprotein sequence coverage. (C) 2011 Elsevier B.V. All rights reserved.