Figure 2.

Quantification of UDP–glucose glycoprotein:glucosyltransferase monoglucosylation of glycopeptide substrates. (A) Monoglucosylation of α-galactosidase (α-Gal) tryptic glycopeptides. Approximately 1 μg of α-Gal was chemically denatured, and was incubated with UDP–glucose glycoprotein:glucosyltransferase (UGGT) at a concentration of ∼25 nM and UDP-³H-glucose at 2.5 μM (19.5 Ci mmol⁻¹). Samples were then either electrophoretically separated by SDS–polyacrylamide gel electrophoresis (lane 1; α-Gal) or cleaved by trypsin, followed by SDS–PAGE (lane 2; labelled first, cut second (L1C2)). In lane 3 (cut first, labelled second (C1L2)), a tryptic digest of α-Gal was incubated with UGGT and 2.5 μM UDP-[³H]glucose (19.5 Ci mmol⁻¹). The gel was exposed for two days, and the resulting autoradiograph is shown. (B) Micro-liquid-chromotography–electrospray mass spectrometry analyses of UGGT-monoglucosylated tryptic glycopeptide T_444–466 from α-Gal (see Fig. 4 for reaction conditions). Two ion chromatograms are shown, representing the full mass spectral acquisition (m/z = 400–2,000) obtained with a low orifice voltage setting (40 V), together with a high orifice voltage scan (100 V) for m/z = 366. (Ba) Mass spectrum from a control tryptic digest of α-Gal with no incubation with UGGT, showing the Man9-GlcNAc2 (m/z = 1,427) and Man10-GlcNAc2 (m/z = 1,481) glycoforms that were used to calculate background levels of molar incorporation. (Bb) Mass spectrum obtained from the incubation of an α-Gal tryptic digest with UGGT and UDP–glucose, showing the Man9-GlcNAc2 (m/z = 1,427) and Glc1-Man9-GlcNAc2 (m/z = 1,481) glycoforms. Peak ratio (molar glucose incorporation) is the (area under the (Glc1 or Man1)-Man9-GlcNAc2 isotopic ion cluster divided by the sum of the areas under the (Glc1 or Man1)-Man9-GlcNAc2 isotopic ion cluster and the area under the Man9-GlcNAc2 isotopic ion cluster. All y axes indicate the ion intensity relative to the highest peak in each spectrum. Rel. int., relative ion intensity.