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Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.

Kudelka MR, Antonopoulos A, Wang Y, Duong DM, Song X, Seyfried NT, Dell A, Haslam SM, Cummings RD, Ju T - Nat. Methods (2015)

Bottom Line: Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS).Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus.Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

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MALDI-TOF-MS and MS/MS profiling of the O-glycome of primary cells. (a,b) CORA was used to profile the O-glycome of HUVECs (a) and primary human dermal fibroblasts (b). 12.5 × 104 (a, b), 5 × 104 (b), and 2.5 × 104 (b) cells were seeded in T25, 6 well, or 12 well flasks, respectively. 50 µM Ac3GalNAc-Bn was added after 2 days, and Bn-O-glycans were purified, permethylated, and analyzed by MALDI after 3 more days. MALDI-TOF/TOF-MS/MS (structure) analysis was performed for HUVECs and MS (composition) was performed for fibroblasts. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Putative structures are based on composition, tandem MS or MS, and biosynthetic knowledge.
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Figure 5: MALDI-TOF-MS and MS/MS profiling of the O-glycome of primary cells. (a,b) CORA was used to profile the O-glycome of HUVECs (a) and primary human dermal fibroblasts (b). 12.5 × 104 (a, b), 5 × 104 (b), and 2.5 × 104 (b) cells were seeded in T25, 6 well, or 12 well flasks, respectively. 50 µM Ac3GalNAc-Bn was added after 2 days, and Bn-O-glycans were purified, permethylated, and analyzed by MALDI after 3 more days. MALDI-TOF/TOF-MS/MS (structure) analysis was performed for HUVECs and MS (composition) was performed for fibroblasts. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Putative structures are based on composition, tandem MS or MS, and biosynthetic knowledge.

Mentions: Next, we evaluated primary human dermal fibroblasts and umbilical vein endothelial cells (HUVECs) (Fig. 5, Supplementary Fig. 18,19). HUVECs produced 43 O-glycan structures, including those containing poly-N-acetyllactosamine, Lewis structures, blood group antigens, and I antigen, as confirmed by MS/MS (Fig. 5a, Supplementary Fig. 18a,19). Fibroblasts also produced at least 18 glycans (unique masses), including poly-N-acetyllactosamine, Lewis structures, and blood group antigens (Fig. 5b, Supplementary Fig. 18b). The remarkable diversity of O-glycans in these cells indicates their potential importance.


Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.

Kudelka MR, Antonopoulos A, Wang Y, Duong DM, Song X, Seyfried NT, Dell A, Haslam SM, Cummings RD, Ju T - Nat. Methods (2015)

MALDI-TOF-MS and MS/MS profiling of the O-glycome of primary cells. (a,b) CORA was used to profile the O-glycome of HUVECs (a) and primary human dermal fibroblasts (b). 12.5 × 104 (a, b), 5 × 104 (b), and 2.5 × 104 (b) cells were seeded in T25, 6 well, or 12 well flasks, respectively. 50 µM Ac3GalNAc-Bn was added after 2 days, and Bn-O-glycans were purified, permethylated, and analyzed by MALDI after 3 more days. MALDI-TOF/TOF-MS/MS (structure) analysis was performed for HUVECs and MS (composition) was performed for fibroblasts. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Putative structures are based on composition, tandem MS or MS, and biosynthetic knowledge.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: MALDI-TOF-MS and MS/MS profiling of the O-glycome of primary cells. (a,b) CORA was used to profile the O-glycome of HUVECs (a) and primary human dermal fibroblasts (b). 12.5 × 104 (a, b), 5 × 104 (b), and 2.5 × 104 (b) cells were seeded in T25, 6 well, or 12 well flasks, respectively. 50 µM Ac3GalNAc-Bn was added after 2 days, and Bn-O-glycans were purified, permethylated, and analyzed by MALDI after 3 more days. MALDI-TOF/TOF-MS/MS (structure) analysis was performed for HUVECs and MS (composition) was performed for fibroblasts. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Putative structures are based on composition, tandem MS or MS, and biosynthetic knowledge.
Mentions: Next, we evaluated primary human dermal fibroblasts and umbilical vein endothelial cells (HUVECs) (Fig. 5, Supplementary Fig. 18,19). HUVECs produced 43 O-glycan structures, including those containing poly-N-acetyllactosamine, Lewis structures, blood group antigens, and I antigen, as confirmed by MS/MS (Fig. 5a, Supplementary Fig. 18a,19). Fibroblasts also produced at least 18 glycans (unique masses), including poly-N-acetyllactosamine, Lewis structures, and blood group antigens (Fig. 5b, Supplementary Fig. 18b). The remarkable diversity of O-glycans in these cells indicates their potential importance.

Bottom Line: Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS).Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus.Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

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