Mass Spectrometric Determination of Site-Specific <i>O</i> -Acetylation in Rhamnogalacturonan I Oligomers

<i>O</i>-acetylation, a common modification in rhamnogalacturonan I (RG-I), is critical for various biological processes, including plant growth, stress responses, and pathogen defense. Precise determination of the degree and specific positions of acetylation is therefore essential. To date, nuclear magnetic resonance (NMR) and tandem mass spectrometry have been employed to identify <i>O</i>-acetyl positions in pectin oligosaccharides. Although NMR is effective, it requires pure, high-concentration samples. Tandem mass spectrometry (MS), which uses smaller sample amounts, faces challenges due to <i>O</i>-acetyl migration between monosaccharide positions. The multiple steps in pectin sample analysis can further promote <i>O</i>-acetyl migration, especially near free hydroxyl groups. Moreover, during tandem MS, <i>O</i>-acetyl groups may detach, complicating the accurate tracking. This study presents an approach to lock <i>O</i>-acetyl groups by introducing trideuteroacetyl and propionyl substituents onto free hydroxyls of RG-I or partially acetylated RG-I. By combining matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and electrospray ionization (ESI) MS with MS/MS or tandem mass spectrometry (MSⁿ), we devised a way to determine the monosaccharide sequence in the oligomer and the precise positions of <i>O</i>-acetyl groups in partially acetylated RG-I. This method enables the study of the regiospecificity of recombinant pectin <i>O</i>-acetyltransferases and can be applied to other oligosaccharides to determine acyl positions.