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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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Sensitivity of CORA. (a) MKN45, (b) Colo205, (c) MDA-MB-231, and (d) MCF-7 cells were seeded at 5 × 105, 105, and 2 × 104 cells/well in 6 well, 12 well, and 48 well flasks. 50 µM Ac3GalNAc-Bn was added and Bn-O-glycans were purified and permethylated after 3 days. 1/150th (6 well, 12 well) or 1/50th (48 well) of total glycans were analyzed by MALDI-MS (composition). (e) O-glycomes were observed from all cells seeded at 5 × 105 and 105 cells/well and 3 of 4 cells seeded at 2 × 104 cells/well. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Representative profiles are shown (n = 2).
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Figure 4: Sensitivity of CORA. (a) MKN45, (b) Colo205, (c) MDA-MB-231, and (d) MCF-7 cells were seeded at 5 × 105, 105, and 2 × 104 cells/well in 6 well, 12 well, and 48 well flasks. 50 µM Ac3GalNAc-Bn was added and Bn-O-glycans were purified and permethylated after 3 days. 1/150th (6 well, 12 well) or 1/50th (48 well) of total glycans were analyzed by MALDI-MS (composition). (e) O-glycomes were observed from all cells seeded at 5 × 105 and 105 cells/well and 3 of 4 cells seeded at 2 × 104 cells/well. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Representative profiles are shown (n = 2).

Mentions: Beta-elimination often requires ≥107 cells and produces many unassignable peaks23. To determine how many cells are needed to get clean, interpretable profiles with CORA, we profiled four cell lines each seeded at 5 × 105, 105, or 2 × 104 cells. We obtained O-glycomes from all lines seeded at ≥105 cells and 3 of 4 lines seeded at 2 × 104 cells (Fig. 4). Notably, profiles did not change with different cell numbers. The detection of Bn-O-glycans from 2 × 104 cells cultured for 3 days (to a total of ~8 × 104 cells assuming ~24 hour doubling times) represents a ~100–1000-fold increase in sensitivity compared to β-elimination.


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)

Sensitivity of CORA. (a) MKN45, (b) Colo205, (c) MDA-MB-231, and (d) MCF-7 cells were seeded at 5 × 105, 105, and 2 × 104 cells/well in 6 well, 12 well, and 48 well flasks. 50 µM Ac3GalNAc-Bn was added and Bn-O-glycans were purified and permethylated after 3 days. 1/150th (6 well, 12 well) or 1/50th (48 well) of total glycans were analyzed by MALDI-MS (composition). (e) O-glycomes were observed from all cells seeded at 5 × 105 and 105 cells/well and 3 of 4 cells seeded at 2 × 104 cells/well. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Representative profiles are shown (n = 2).
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Related In: Results  -  Collection

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Figure 4: Sensitivity of CORA. (a) MKN45, (b) Colo205, (c) MDA-MB-231, and (d) MCF-7 cells were seeded at 5 × 105, 105, and 2 × 104 cells/well in 6 well, 12 well, and 48 well flasks. 50 µM Ac3GalNAc-Bn was added and Bn-O-glycans were purified and permethylated after 3 days. 1/150th (6 well, 12 well) or 1/50th (48 well) of total glycans were analyzed by MALDI-MS (composition). (e) O-glycomes were observed from all cells seeded at 5 × 105 and 105 cells/well and 3 of 4 cells seeded at 2 × 104 cells/well. Spectra are off-set for each seeding density and scaled relative to maximum intensity. Representative profiles are shown (n = 2).
Mentions: Beta-elimination often requires ≥107 cells and produces many unassignable peaks23. To determine how many cells are needed to get clean, interpretable profiles with CORA, we profiled four cell lines each seeded at 5 × 105, 105, or 2 × 104 cells. We obtained O-glycomes from all lines seeded at ≥105 cells and 3 of 4 lines seeded at 2 × 104 cells (Fig. 4). Notably, profiles did not change with different cell numbers. The detection of Bn-O-glycans from 2 × 104 cells cultured for 3 days (to a total of ~8 × 104 cells assuming ~24 hour doubling times) represents a ~100–1000-fold increase in sensitivity compared to β-elimination.

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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