Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, Pennsylvania 29425, United States.
J Proteome Res. 2022 Aug 5;21(8):1930-1938. doi: 10.1021/acs.jproteome.2c00193. Epub 2022 Jun 29.
Alterations to N-glycan expression are relevant to the progression of various diseases, particularly cancer. In many cases, specific N-glycan structural features such as sialylation, fucosylation, and branching are of specific interest. A novel MALDI imaging mass spectrometry workflow has been recently developed to analyze these features of N-glycosylation through the utilization of endoglycosidase enzymes to cleave N-glycans from associated glycoproteins. Enzymes that have previously been utilized to cleave N-glycans include peptide-N-glycosidase F (PNGase F) to target N-glycans indiscriminately and endoglycosidase F3 (Endo F3) to target core fucosylated N-glycans. In addition to these endoglycosidases, additional N-glycan cleaving enzymes could be used to target specific structural features. Sialidases, also termed neuraminidases, are a family of enzymes that remove terminal sialic acids from glycoconjugates. This work aims to utilize sialidase, in conjunction with PNGase F/Endo F3, to enzymatically remove sialic acids from N-glycans in an effort to increase sensitivity for nonsialylated N-glycan MALDI-IMS peaks. Improving detection of nonsialylated N-glycans allows for a more thorough analysis of specific structural features such as fucosylation or branching, particularly of low abundant structures. Sialidase utilization in MALDI-IMS dramatically increases sensitivity and increases on-tissue endoglycosidase efficiency, making it a very useful companion technique to specifically detect nonsialylated N-glycans.
糖基化修饰的改变与各种疾病的进展有关，尤其是癌症。在许多情况下，特定的 N-糖链结构特征，如唾液酸化、岩藻糖化和分支，具有特定的研究意义。最近开发了一种新型 MALDI 成像质谱工作流程，通过使用内切糖苷酶从相关糖蛋白中切割 N-聚糖，来分析 N-糖基化的这些特征。以前用于切割 N-聚糖的酶包括肽-N-糖苷酶 F（PNGase F），以非特异性地靶向 N-聚糖，和内切糖苷酶 F3（Endo F3），以靶向核心岩藻糖化的 N-聚糖。除了这些内切糖苷酶外，还可以使用其他 N-聚糖裂解酶来靶向特定的结构特征。唾液酸酶，也称为神经氨酸酶，是一组从糖缀合物中去除末端唾液酸的酶。本工作旨在利用唾液酸酶与 PNGase F/Endo F3 联合使用，从 N-聚糖中酶切唾液酸，以提高非唾液酸化 N-聚糖 MALDI-IMS 峰的灵敏度。提高非唾液酸化 N-聚糖的检测灵敏度可以更全面地分析岩藻糖化或分支等特定结构特征，尤其是低丰度结构。唾液酸酶在 MALDI-IMS 中的应用极大地提高了灵敏度，并提高了组织内内切糖苷酶的效率，使其成为一种非常有用的辅助技术，可专门检测非唾液酸化的 N-聚糖。
