Chitosan-Functionalized Magnetic Graphene Oxide Nanocomposites Enable High-Purity mRNA Enrichment To Avoid False Positives in LC-MS/MS Modification Profiling

Messenger RNA (mRNA) has emerged as a transformative therapeutic frontier, and the discovery of its chemical modifications has revealed critical post-transcriptional regulatory mechanisms. Accurate investigation of these modifications requires ultrapure mRNA, yet conventional isolation methods often suffer from rRNA contamination. To address this issue, we developed chitosan-functionalized magnetic graphene oxide nanocomposites (Fe 3 O 4 /GO/CS) engineered with tailored surface properties. The nanocomposites leverage chitosan for steric hindrance against magnetic aggregation and graphene oxide for its ultrahigh surface area, enabling high-density oligo(dT) probe immobilization. The platform achieved specific mRNA capture with markedly reduced rRNA coisolation since the optimized negative zeta potential and surface chemistry preferentially capture linear poly(A)-tailed mRNA over structured rRNA through differential electrostatic and π–π stacking interactions. When applied to total RNA and mitochondrial RNA from PC12 cells, Fe 3 O 4 /GO/CS demonstrated 1.5-fold higher mRNA enrichment efficiency compared with commercial kits. Comprehensive validation of the enriched mRNA using a bioanalyzer and agarose gel electrophoresis confirmed a significant reduction of 18S and 28S rRNA contamination by Fe 3 O 4 /GO/CS, with residual levels decreasing to below 1%. The resulting high-purity mRNA enabled sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) modification profiling, allowing for accurate quantification of low-abundance mRNA modifications. Additionally, background interference from rRNA modifications (such as 2’- O -methylation) and specific oligonucleotide fragments was significantly reduced, substantially avoiding the risk of false-positive identifications. This novel platform established an effective sample preparation method for mRNA modification analysis, while its expandability to diverse downstream applications provides high-quality mRNA for empowering next-generation mRNA-based diagnostics and therapeutics.