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Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation.
ACS Chemical Biology ( IF 4 ) Pub Date : 2020-08-18 , DOI: 10.1021/acschembio.0c00453
Asif Shajahan 1 , Nitin T Supekar 1 , Han Wu 2 , Amberlyn M Wands 2 , Ganapati Bhat 1 , Aravind Kalimurthy 1 , Masaaki Matsubara 1 , Rene Ranzinger 1 , Jennifer J Kohler 2 , Parastoo Azadi 1
Affiliation  

Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.

中文翻译:

用于细胞动态糖基化明确分析的质谱方法。

细胞水平的各种生物过程受糖基化调节,糖基化是蛋白质和脂质上高度微异质的翻译后修饰 (PTM)。可以通过将非天然糖代谢并入聚糖表位并使用生物正交探针检测它们来研究糖基化的动态性质。然而,由于非特异性背景反应和非天然糖代谢的模糊性,这种方法具有显着的缺点。在这里,我们报告了使用质谱 (MS) 通过糖蛋白质组学和糖组学直接检测它们的无探针策略。该方法显着简化了通过混杂唾液酸、N-乙酰-d安装的带有单糖的非天然官能团的检测-半乳糖胺(GalNAc)和N-乙酰-d-葡萄糖胺 (GlcNAc) 生物合成途径。通过细胞分馏、随后的基于 ZIC-HILIC(两性离子-亲水相互作用色谱)的糖肽富集以及用于 MS 数据处理的光谱富集算法对糖蛋白进行多阶段富集,从而能够直接检测在 N 上以低丰度掺入的非天然单糖/O-糖肽及其天然对应物。我们的方法允许检测天然和非天然含糖糖肽、N-和 O-糖肽、聚糖上非天然单糖类型的区分以及它们通过定量的掺入效率。通过这一点,我们可以推断单糖在加工过程中通过聚糖回收途径和随后掺入聚糖链中的相互转化。
更新日期:2020-10-17
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