Unraveling O-Glycan Diversity of Mucins: Insights from SmE Mucinase and Ultraviolet Photodissociation Mass Spectrometry

Abstract

Deciphering the pattern and abundance of O-glycosylation of mucin domain proteins, glycoproteins heavily implicated in cancer and other diseases, remains an ongoing challenge. Both the macro- and microheterogeneity of glycosylation complicates the analysis, motivating the development of new strategies for structural characterization of this diverse class of glycoproteins. Here we combine digestion of mucin domain proteins using a targeted protease, Enhancin from Serratia marcescens (SmE), with ultraviolet photodissociation (UVPD) mass spectrometry to advance glycan mapping and elucidation of O-glycosylation trends of densely glycosylated mucin proteins. UVPD facilitates identification of O-glycoforms of mucin domain proteins TIM-1, MUC-1 and MUC-16. Additionally, UVPD elucidates several glycoforms of MUC-16 and contributes to the discovery of O-glycosylation across tandem repeats of MUC-1.