The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment
The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment
复制标题
DOI：
10.1074/mcp.m113.027953
复制
发表时间：
2013-10-01
影响因子：
7
通讯作者：
Lee, Richard S.
中科院分区：
文献类型：
--
作者：
Zhou, Hui;Froehlich, John W.;Lee, Richard S.
目前研究复杂样品中N-糖蛋白的策略通常是离散的，集中于通过基于糖的技术富集的N-聚糖或N-糖基。在这项研究中，我们报告了一个简单快速的样品制备平台，GlycoFilter，它允许在一个工作流程中全面表征N-聚糖，N-糖基和蛋白质。PNGase F催化的去N-糖基化和胰蛋白酶消化均通过微波辐射加速，并在单个旋转过滤器中依次进行。通过过滤分别收集N-聚糖和肽（包括去N-糖基化肽）。在模型糖蛋白、牛核糖核酸酶B、牛胎球蛋白和人血清IgG上建立了有效收集复杂和异质N-聚糖的条件。利用该平台对人尿液和血浆中的N-糖组、N-糖蛋白组和蛋白质组进行了表征。总体而言，从三对尿液和血浆样本中分别鉴定出共计865个和295个N-糖基。许多位点被明确定义为通过检测其非糖修饰肽部分占据（128个来自尿液，61个来自血浆），表明N-糖基化的部分占据经常发生。考虑到部分占据的可能高流行率和可变性，仅基于去糖基化肽的糖蛋白定量可能导致不准确的定量。
Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.