Novel Combined Enzymatic Approach to Analyze Nonsialylated N-Linked Glycans through MALDI Imaging Mass Spectrometry

Abstract

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.