can be generated and harvested from MS/MS spectra of the resulting glycopeptides. Several Oglycoproteases have been explored for generating O-glycopeptides, and a few, aptly named mucinases, are applicable to the extremely complex mucin-type O-glycoproteins. Mucinases can navigate the highly glycosylated regions of mucin glycoproteins and cleave between adjacent glycosites, resulting in small peptides that can contain multiple occupied glycosites. Early discovered mucinases include StcE from E. coli and BT4244 from Bacteroides thetaiotaomicron. BT4244 has been used previously to generate O-glycopeptides for analysis by mass spectrometry (MS), and its specificity for O-glycoproteins has been examined. Similarly, StcE has been used to map O-glycosites on mucin proteins and to investigate the "bottlebrush" structure of mucin domains. A newly introduced mucinase, SmE from Serratia marcescens, has been utilized for characterizing the complex TIM family of mucin domain proteins, whose glycosylation patterns have not been fully cataloged. The utility of SmE for the study of highly glycosylated regions is especially pivotal for examination of mucin proteins, such as the human mucin proteins expressed by epithelial cells in the respiratory, digestive, gastrointestinal, and urogenital tracts (MUC1-22) that are implicated in various diseases. Each mucin protein is complex, and notably the aberrant glycosylation of several MUC proteins has been correlated with different cancers, an outcome that could be related to the overexpression or deregulation of the MUC proteins. With its proficient ability to cleave between adjacent glycosites, SmE is poised to expand characterization of the most complex glycoproteins.