Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
J Proteome Res. 2012 Mar 2;11(3):1949-57. doi: 10.1021/pr2011268. Epub 2012 Feb 22.
N-Linked glycoproteins are involved in several diseases and are important as potential diagnostic molecules for biomarker discovery. Therefore, it is important to provide sensitive and reliable analytical methods to identify not only the glycoproteins but also the sites of glycosylation. Recently, numerous strategies to identify N-linked glycosylation sites have been described. These strategies have been applied to cell lines and several tissues with the aim of identifying many hundreds (or thousands) of glycosylation events. With high-throughput strategies however, there is always the potential for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyzes the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site. We have evaluated this common large-scale N-linked glycoproteomic strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery found in mammalian systems and some pathogenic microbes. After isolation and proteolytic digestion of E. coli membrane proteins, we investigated the presence of deamidated asparagines. The results show the presence of deamidated asparagines especially with close proximity to a glycine residue or other small amino acid, as previously described for spontaneous in vivo deamidation. Moreover, we have identified deamidated peptides with incorporation of (18)O, showing the pitfalls of glycosylation site assignment based on deamidation of asparagine induced by PNGase F in (18)O-water in large-scale analyses. These data experimentally prove the need for more caution in assigning glycosylation sites and "new" N-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Besides showing the spontaneous deamidation, we provide alternative methods for validation that should be used in such experiments.
N-连接糖蛋白参与多种疾病，并且作为生物标志物发现的潜在诊断分子非常重要。因此，提供敏感可靠的分析方法来鉴定不仅糖蛋白，而且糖基化位点是很重要的。最近，已经描述了许多鉴定 N-连接糖基化位点的策略。这些策略已应用于细胞系和几种组织，旨在鉴定数百（或数千）个糖基化事件。然而，使用高通量策略时，总是存在假阳性的可能性。这种混淆源于用于将 N-聚糖从先前糖基化肽中分离出来的蛋白 N-糖苷酶 F（PNGase F）反应，它催化裂解并脱酰胺天冬酰胺残基。这通常被认为是有益的，因为它突出了修饰位点。我们评估了这种常见的大规模 N-连接糖蛋白质组学策略，并使用大肠杆菌作为模型生物证明了潜在的陷阱，因为它缺乏哺乳动物系统和一些致病性微生物中发现的 N-糖基化机制。在分离和蛋白水解消化大肠杆菌膜蛋白后，我们研究了脱酰胺天冬酰胺的存在。结果表明，特别是在靠近甘氨酸残基或其他小氨基酸的情况下，存在脱酰胺天冬酰胺，如先前描述的自发体内脱酰胺。此外，我们已经鉴定了具有（18）O 掺入的脱酰胺肽，表明在大规模分析中基于 PNGase F 在（18）O 水中诱导的天冬酰胺脱酰胺来分配糖基化位点存在陷阱。这些数据实验证明，在没有适当对照实验的情况下，基于常见的 N-连接糖蛋白质组学策略分配糖基化位点和“新”N-连接共识位点需要更加谨慎。除了显示自发脱酰胺外，我们还提供了替代验证方法，应在这种实验中使用。
