A precise and versatile platform for rapid glycosylation analysis of brain tissue,Analytical Methods

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A precise and versatile platform for rapid glycosylation analysis of brain tissue
Analytical Methods ( IF 2.7 ) Pub Date : 2020-02-28 , DOI: 10.1039/c9ay02596k
Mattia Vacchini _{1,

2,

3,

4,

5} , Laura Cipolla _{4,

5,

6,

7} , Olga Gornik _{2,

3,

8,

9} , Gordan Lauc _{1,

2,

3,

8,

9} , Thomas Klarić _{2,

3,

8,

9}

Affiliation

Glycans influence a variety of aspects of tissue development and pathophysiology, harboring a wealth of biochemical information exploitable for the discovery of new biomarkers. The development of versatile and precise glycoanalytical platforms is crucial to bring to the scientific community reliable tools to unveil the glycan-encoded biochemical information. In this work, a novel platform for brain glycan analysis, named Lysate in-Solution Deglycosylation (LSD), is presented, validated and compared to similar methodologies reported in the literature. Briefly, within the LSD workflow, brain tissue is lysed, proteins are purified via methanol/chloroform extraction, and directly deglycosylated with PNGase F. Released N-glycans are purified using a second round of methanol/chloroform extraction, labelled with fluorescent tags, and cleaned-up prior to LC-FLR/MS analysis. LSD is a quick, sensitive, robust, precise, specific and versatile glycomic workflow, compatible with multiple glyco(proteo)mics analyses of the same sample and eventually applicable to other solid tissues. LSD was devised as a plug-and-play platform for quick glycosylation analysis, and was designed to be compatible with a variety of analytical set-ups (i.e., diverse detection systems), thus enabling the extraction of an additional layer of information from different studies (i.e., proteomics) without the need to collect fresh biological materials. In comparison to analogous N-glycomic workflows, LSD performs reliably on the smallest amount of starting brain tissue and, to the best of the authors' knowledge, is currently the most thoroughly validated neuro-N-glycomic method in the field-related literature, thus representing a significant step forward on the road towards glycan-based biomedical applications.

中文翻译：

快速，精确的大脑组织糖基化分析平台

聚糖影响组织发育和病理生理学的各个方面，具有丰富的生化信息可用于发现新的生物标记。通用且精确的糖分析平台的开发对于为科学界带来可靠的工具以揭示聚糖编码的生化信息至关重要。在这项工作中，提出了一种新型的用于脑聚糖分析的平台，称为溶血素溶液中去糖基化（LSD），并与文献中报道的类似方法进行了比较。简言之，将LSD工作流内，脑组织被裂解，纯化蛋白质通过甲醇/氯仿提取，并直接用PNGase F.发行日期去糖基化Ñ使用第二轮甲醇/氯仿萃取纯化β-聚糖，并用荧光标签标记，然后在LC-FLR / MS分析之前进行纯化。LSD是一种快速，灵敏，稳健，精确，特异和通用的糖组学工作流程，可与同一样品的多种糖组学分析兼容，并最终适用于其他实体组织。LSD被设计为用于快速糖基化分析的即插即用平台，并被设计为与各种分析设置（即，各种检测系统）兼容，从而能够从不同的位置提取附加的信息层。研究（即蛋白质组学）而无需收集新鲜的生物材料。与类似的N相比LSD工作流程中，LSD在最小数量的起始脑组织上运行可靠，据作者所知，LSD是目前在相关领域文献中得到最充分验证的神经N血糖方法，因此代表了重要的一步在基于聚糖的生物医学应用的道路上前进。

更新日期：2020-02-28

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