Simple One-Step Deuterated Peracetylation-MALDI-MS/MS (OSDPM) Strategy for Structural Sequencing of Glycosaminoglycan...

Heparin and heparan sulfate (HP/HS), characterized by heterogeneous sulfation motifs, uronic acid epimerization (GlcA/IdoA), and variations in N-acetyl and N-sulfo groups, play pivotal roles in regulating physiological and pathological processes. This inherent structural complexity, resulting from sulfation heterogeneity and epimeric diversity, poses significant challenges for the HP/HS structural analysis. Current labor-intensive multistep derivatization methods coupled with LC-MS/MS or MSⁿ usually require a substantial amount of sample (>50 μg) and prolonged processing times, which severely limit their applicability to scarce HP/HS samples. Herein, we develop a one-step deuterated peracetylation-MALDI-MS/MS (OSDPM) analytical strategy that enables high-throughput structural characterization of HP/HS oligosaccharides with ng- to sub-μg-level sensitivity. Additionally, this OSDPM approach simultaneously determines the oligosaccharide composition and sulfation patterns and distinguishes between GlcA2S/IdoA2S epimers. We successfully applied this method to characterize the structures of 17 natural HP/HS glycans separated from porcine intestinal mucosa, and their glycan microarray data further validated the structural accuracy of OSDPM-derived sequencing results. The simplicity, sensitivity, and efficiency of our OSDPM strategy provide a valuable solution for HP/HS structural characterization.