Specific isolation of O-linked N-acetylglucosamine glycopeptides from complex mixtures.
Galactosyltransferase and UDP-[3H]galactose are commonly used to identify O-linked N-acetylglucosamine (O-GlcNAc)-bearing proteins and peptides. In this report we show that immobilized Ricinus communis agglutinin I (RCA I) specifically binds in vitro galactosylated O-GlcNAc-bearing peptides, facilitating their selective isolation from complex mixtures. First, the peptide YSDSPSTST was O-GlcNAc glycosylated, galactosylated, and sialylated. Of these three glycoforms, only the one with a terminal galactose interacted with the lectin. Next, RCA I was used to isolate glycopeptides from the O-GlcNAc-bearing basic phosphoprotein (BPP) of human cytomegalovirus. BPP was overexpressed using baculovirus, [3H]galactosylated, digested with trypsin, and fractionated on RCA I. Peptides that were not galactosylated passed through the column, whereas the majority of the radiolabeled glycopeptides interacted weakly with the lectin and did not require lactose or elution. These radiolabeled peptides eluted as a broad peak with the leading edge being characterized by more hydrophobic glycopeptides and the lagging edge by less hydrophobic peptides, suggesting that the polypeptide backbone may influence the interaction with the lectin. Lactose was required to elute the remaining radiolabeled peptides, suggesting that these peptides are multiply glycosylated. The weakly interacting glycopeptides were analyzed directly by liquid chromatography/electrospray-mass spectrometry (LC/ES-MS). Glycopeptides corresponding to both of the major sites of glycosylation of BPP were identified. Thus, RCA I greatly facilitates the selective isolation of in vitro galactosylated O-GlcNAc glycopeptides from complex mixtures and substantially reduces the purification required for subsequent site-mapping by gas-phase sequencing and/or LC/ES-MS.
Hayes
B K
BK
Department of Biochemistry and Molecular Genetics, School of Medicine and Dentistry, University of Alabama at Birmingham 35294-0005, USA.
Greis
K D
KD
Hart
G W
GW
D000117:Acetylglucosamine / Q000737:chemistry / Q000302:isolation & purification*; D000373:Agglutinins; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D012278:Ricinus / Q000737:chemistry
D000373:Agglutinins; D006020:Glycopeptides; D000117:Acetylglucosamine