PMC

(A) Glycoproteins containing terminal GlcNAc were purified by first treating N2 larval lysates with both UDP-azido-GalNAc and GalT (+), or with UDP-azido-GalNAc alone (−), then reacting with biotin-alkyne, and finally capturing the biotinylated glycoproteins with streptavidin beads. Biotinylated glycoproteins were visualized by Western blot with avidin:HRP. (B) Eluted glycoproteins were purified as in (A), fractionated by SDS-PAGE, and the gel was stained with Sypro. Peptides shared by ACT-2, ACT-3, and ACT-4 were identified by MALDI-TOF/TOF from the asterisk-marked band, which was exclusive to the (+) lane.

(A) During copper-catalyzed azide-alkyne cycloaddition (“Click Chemistry”), a covalent bond is formed between a terminal alkyne and an azide group. This reaction can be used to covalently label glycoproteins with biotin or fluorescent tags. This occurs when the R₁ group attached to the azide is a monosaccharide linked to a glycoprotein and the R₂ group attached to the alkyne is biotin or a fluorophore (e.g. TAMRA). (B) N2 cells were cultured with 40 µM GalNAc (−) or 40 µM azido-GalNAc (+) and cell lysates were reacted with TAMRA-alkyne by Click Chemistry. Metabolically labeled proteins were detected by TAMRA fluorescence, and protein loading was verified by Coomassie gel staining. (C) N2 cells were cultured with 40 µM GlcNAc (−) or 40 µM azido-GlcNAc (+) and cell lysates were reacted with biotin-alkyne by Click Chemistry. Metabolically labeled proteins were detected by avidin-HRP with AEC (colorimetric detection), and protein loading was verified by staining with the fluorescent protein stain, Sypro. (D) Conditioned media collected from primary embryonic N2 cell cultures grown without serum in the presence of 40 µM GalNAc (−) or 40 µM azido-GalNAc (+) was reacted with biotin-alkyne. Metabolically labeled secreted proteins were detected by avidin-HRP with ECL, and protein loading was verified by Sypro staining. Asterisk in (C) and (D) marks a known 83 kDa endogenous avidin-binding protein .

(A) Glycoproteins were purified from bovine heart extracts enriched for mitochondria by incubating the detergent-solubilized proteins with Wheat Germ Agglutinin (WGA) agarose beads. After extensive PBS washing, the captured glycoproteins were specifically eluted with PBS supplemented with 0.5 M GlcNAc, which is a monosaccharide that competes with glycoproteins for the sugar-binding sites on the WGA-beads. Input and non-binding flowthrough (FT) lanes contain 0.03% of each fraction. The entirety of the last wash and the eluate fractions and were precipitated, resuspended in Laemmli buffer, and loaded into their respective gel lanes. ATP synthase α-subunit was detected by western blot. (B) Bovine heart extracts enriched for mitochondria were immunoprecipitated with an ATP synthase Complex V capture antibody. Input, FT, and eluate fractions were loaded in duplicate lanes. One half of the blot was probed with WGA:HRP, then stripped and probed with ATP synthase β-subunit antibody; the other half of the blot was probed with ATP synthase α-subunit antibody. Representative Sypro staining from the WGA-probed blot half is shown. (C) COS-7 cells were incubated with 50 µM azido-GalNAc (+) or 50 µM GalNAc (−). Lysates were reacted with biotin-alkyne, and biotinylated azido-labeled proteins were captured with streptavidin beads. Input and FT lanes contain 2.5% of sample; eluate lanes, 50%. Azido-GalNAc and GalNAc samples were always handled in parallel.

C. elegans embryonic cells grown with 40 µM azido-GalNAc (top panels) or 40 µM GalNAc (bottom panels) were reacted with TAMRA-alkyne and fractionated by 2DE. (A), (B), and (C) each show a different field of view representative of two independent experiments. Within each field of view, metabolically labeled glycoproteins were detected in-gel by their TAMRA fluorescence (left panels). And subsequently, the total protein profile of the sample was visualized with Sypro protein stain (right panels). Arrowheads mark glycoprotein candidates that met the criteria listed in . Some spots marked with arrowheads were cut from the gels shown while others were cut from the gels of the other independent experiment. MALDI-TOF/TOF identified the following C. elegans proteins within each arrowhead-marked spot: (a) ATP synthase α-subunit (H28O16.1); (b) Acyl CoA Dehydrogenase family member (acdh-12); (c) Ubiquinol-Cytochrome c oxidoreductase complex family member (ucr-2.3); (d) Actin (peptides shared by ACT-2, ACT-3, and ACT-4 were observed); (e) TCTP (translationally-controlled tumor protein) homolog family member (tct-1); (f) Aspartyl protease family member (asp-4); (g) Tubulin, Beta family member (tbb-1); (h) Tubulin, Beta family member (tbb-1). TBB-1 was found in two different 2DE spots likely due to post-translational modification or proteolytic processing.