A Broad-Specificity -Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex -Glycans.

Characterization of mucin-type -glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several -glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known -glycoproteases have specificity constraints, such as inefficient cleavage of glycoproteins bearing sialylated -glycans, high selectivity for certain types of glycoproteins, or protein sequence bias. These factors limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from . Peptide sequence selectivity and its impact on IMPa activity were interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent -glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an -glycosite were cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated -glycans of varying complexity. We present the use of IMPa in a one-step -glycoproteomic workflow for glycoprofiling of the purified glycoproteins granulocyte colony-stimulating factor and receptor-type tyrosine-protein phosphatase C without the need for glycopeptide enrichment. In these examples, IMPa enabled both the identification of -glycosites and the range of complex -glycan structures at each site.