Precision Characterization of Site-Specific O‑Acetylated Sialic Acids on N‑Glycoproteins

O-Acetylation is a common modification of sialic acid, playing a significant role in glycoprotein stability, immune response, and cell development. Due to the lack of efficient methods for direct analysis of O-acetylated sialoglycopeptides (O-AcSGPs), the majority of identified O-acetylated sialic acids (O-AcSia) until now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of O-AcSGPs with probability estimation by recognizing the characteristic B and Y ions of O-AcSias. With further optimization of mass spectrometry parameters, the method allowed us to identify a total of 171 unique O-AcSGPs in mouse serum. Although the majority of these O-AcSGPs were at a relatively low abundance compared with their non-O-acetylated states, they were mainly involved in peptidase/endopeptidase inhibitor activities. The method paves the way for large-scale structural and functional analyses of site-specific O-AcSias in various complex samples as well as further identification of many other similar chemical modifications on glycoproteins.