O-glycan Site-specific Digestion of EPO

Application

Simple and direct O-glycan site identification by site-specific digestion, generating fragments which can be used for high-resolution middle-level analysis.

With simpler substrate proteins carrying only one or a few O-glycosylation sites OpeRATOR can be used to map O-glycosylation sites at the middle-level. Erythropoietin (EPO) is a ~30 kDa glycoprotein with a single O-glycosylation site. After removal of N-glycans by PNGase F and desialylation using SialEXO, the native protein was digested with OpeRATOR and the resulting protein fragments analyzed by reverse phase LC-MS.

Because EPO contains only one O-glycosylation site, the localization of the O-glycosylation can be inferred directly from identifying the site of OpeRATOR digestion. As expected, digestion with OpeRATOR and SialEXO generated two fragments. Analysis of the fragments confirms O-glycosylation at serine residue 126, with the second fragment being unglycosylated.

Site-specific digestion for identification of O-glycan site

The efficiency of OpeRATOR digestion is clear as there is only a very minor portion of undigested EPO which can be identified. The example here highlights how OpeRATOR efficiently digests at sites of O-glycosylation for glycan site mapping; however, it also shows how this enzyme can be used to perform middle-level analysis of some O-glycosylated proteins. Because digestion occurs only at the site of O-glycosylation, for proteins containing only one or a few O-glycan sites, this is an elegant solution to digest these proteins into smaller portions for higher resolution, middle-level analysis which has a number of advantages over intact analysis, especially for complex glycoproteins.

Middle-level characterization of EPO by digestion at the O-glycan site

Specific digestion N-terminally of the O-glycosylation site. Erythropoietin (EPO) was first treated with PNGase F, to remove N-glycans, before digestion using OpeRATOR and SialEXO. The reduced fragments were separated on a reversed phase C4 column followed by ESI-QTOF MS detection. The EPO protein carrying one core 1 O-glycan was hydrolyzed at a single specific site N-terminally of the O-glycosylated serine.

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