SPECIFIC ISOLATION OF O-LINKED N-ACETYLGLUCOSAMINE GLYCOPEPTIDES FROMCOMPLEX-MIXTURES

Galactosyltransferase and UDP-[H-3]galactose are commonly used to iden tify O-linked N-acetylglucosamine (O-GlcNAc)-bearing proteins and pept ides. In this report we show that immobilized Ricinus communis aggluti nin I (RCA I) specifically binds in vitro galactosylated O-GlcNAc-bear ing peptides, facilitating their selective isolation from complex mixt ures. First, the peptide YSDSPSTST was O-GlcNAc glycosylated, galactos ylated, and sialylated. Of these three glycoforms, only the one with a terminal galactose interacted with the lectin. Next, RCA I was used t o isolate glycopeptides from the O-GlcNAc-bearing basic phosphoprotein (BPP) of human cytomegalovirus. BPP was overexpressed using baculovir us, [H-3]galactosylated, digested with trypsin, and fractionated on RC A I. Peptides that were not galactosylated passed through the column, whereas the majority of the radiolabeled glycopeptides interacted weak ly with the lectin and did not require lactose for elution. These radi olabeled peptides eluted as a broad peak with the leading edge being c haracterized by more hydrophobic glycopeptides and the lagging edge by less hydrophobic peptides, suggesting that the polypeptide backbone m ay influence the interaction with the lectin. Lactose was required to elute the remaining radiolabeled peptides, suggesting that these pepti des are multiply glycosylated. The weakly interacting glycopeptides we re analyzed directly by liquid chromatography/electrospray-mass spectr ometry (LC/ES-MS). Glycopeptides corresponding to both of the major si tes of glycosylation of BPP were identified. Thus, RCA I greatly facil itates the selective isolation of in vitro galactosylated O-GlcNAc gly copeptides from complex mixtures and substantially reduces the purific ation required for subsequent site-mapping by gas-phase sequencing and /or LC/ES-MS. (C) 1995 Academic Press, Inc.