Precision Characterization of Site-Specific -Acetylated Sialic Acids on -Glycoproteins.

-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 -acetylated sialoglycopeptides (-AcSGPs), the majority of identified -acetylated sialic acids (-AcSia) until now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of -AcSGPs with probability estimation by recognizing the characteristic B and Y ions of -AcSias. With further optimization of mass spectrometry parameters, the method allowed us to identify a total of 171 unique -AcSGPs in mouse serum. Although the majority of these -AcSGPs were at a relatively low abundance compared with their non--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 -AcSias in various complex samples as well as further identification of many other similar chemical modifications on glycoproteins.