One-Pot <i>O</i> -Glycan Release and Permethylation (OPORP) Enables Rapid and Quantitative <i>O</i> -Glycan Analysis from Low-Input Samples

<i>O</i>-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 <i>O</i>-glycosidase make simple and reproducible <i>O</i>-glycan analysis a long-standing challenge, especially for low-input samples. Current <i>O</i>-glycan preparation workflows typically require microgram-level starting protein material and involve laborious derivatization and purification steps. Moreover, many <i>O</i>-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 <i>O</i>-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 <i>O</i>-glycan profiling from nanogram-level protein samples within 2 h. Notably, major <i>O</i>-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 <i>O</i>-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 <i>O</i>-glycan analysis with broad applications.