Analysis of N-Glycans Released from Glycosylated RNAs

Glycosylated RNAs (glycoRNAs) are involved in immune response, cellular function, gene regulation, and disease development. We report a liquid chromatography-mass spectrometry (LC-MS) method to analyze glycans released from glycoRNAs. The developed method includes extracting RNA from mouse liver using TRIzol, digesting proteins with proteinase K under denaturing conditions, re-extracting RNA using TRIzol, releasing N-glycans by enzymatic digestion, enriching glycans by solid-phase extraction, and analyzing glycans by LC-MS. The method was optimized to detect low-abundance N-glycans from a purchased mixture of N-glycan standards, and 65 glycans were identified. We then applied the method to analyze the glycans released from the glycoRNAs in mouse liver and identified 244 N-glycans from triplicate analyses. We also detected 40 significantly changed N-glycans in the livers of 32-week-old female and male KvB2 mice. This study establishes a simple and efficient technique for characterizing N-glycans in glycoRNAs and paves the way for future studies to understand the function of glycoRNAs.