One-Pot O-Glycan Release and Permethylation (OPORP) Enables Rapid and Quantitative O-Glycan Analysis from Low-Input...

O-glycosylation, an exceptionally complex and heterogeneous post-translational modification, plays pivotal roles in diverse biological and pathological processes, and is a key regulator of biopharmaceutical quality and efficacy. However, the vast structural diversity and the absence of a universal O-glycosidase make simple and reproducible O-glycan analysis a long-standing challenge, especially for low-input samples. Current O-glycan preparation workflows typically require microgram-level starting protein material and involve laborious derivatization and purification steps. Moreover, many O-glycan release methods are prone to "peeling" reactions, leading to glycan degradation and compromised quantitative accuracy. Here, we present a highly efficient One-Pot strategy for simultaneous O-glycan Release and Permethylation, termed OPORP, yielding derivatized glycans compatible with both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and widely available reversed-phase liquid chromatography-mass spectrometry (RPLC-MS). Integrated MALDI-MS, OPORP enables comprehensive O-glycan profiling from nanogram-level protein samples within 2 h. Notably, major O-glycans could be detected from as low as 1 ng of fetuin input and as few as 1,000 MCF-7 cells using RPLC-MS. The method also provides low inter- and intra-assay variability (CV < 20%) and good quantitative linearity for low-input samples (R² ≥ 0.95). With robust quantitative performance, we reveal markedly distinct O-glycan profiles between darbepoetin alfa and a higher-potency novel analog with accuracy. Overall, the simple yet powerful OPORP strategy combines exceptional sensitivity, throughput, and robust quantification, establishing a new methodological benchmark for O-glycan analysis with broad applications.