Imaging the Sialome during Zebrafish Development with Copper‐Free Click Chemistry

Dehnert, Karen W.; Baskin, Jeremy M.; Laughlin, Scott T.; Beahm, Brendan J.; Naidu, Natasha; Amacher, Sharon L.; Bertozzi, Carolyn R.

doi:10.1002/cbic.201100649

Cited by 85 publications

(71 citation statements)

References 31 publications

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“…In particular, exploiting the cellular metabolism of an organism with azide-modified sugars has demonstrated a variety of experimental opportunities both for N-linked [32] and O-linked glycans [57,58]. Furthermore, the ability to provide real-time pulse-chase observations in living organisms such as the zebrafish [31,33,34] has been possible. Recent studies have also demonstrated the characterization of glycopeptides at the proteome level using bioorthogonal sugar residues [37,38].…”

Section: Resultsmentioning

confidence: 98%

“…Examples include bacterial cell surface labeling of using copper(I)-catalyzed [3+2] cycloaddition [28] and site-specific insertion of a bioorthogonal amino acid into a protein sequence [29]. Bertozzi et al [30] were the first to use copper-free click chemistry to link a fluorescent label to a bioorthogonal azido group incorporated metabolically onto protein glycans, and this methodology was subsequently used in many imaging studies (e.g., [31][32][33][34]). The sialic acid pathway is of most interest due to many of the aforementioned properties regarding its role in mAbs.…”

Section: Introductionmentioning

confidence: 98%

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Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

et al. 2015

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Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.

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Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 98%

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

et al. 2015

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“…Then, ca . 1.0 × 10 6 cells were harvested from each culture, washed, and treated with 2 M acetic acid at 80 °C for 2 h to release sialic acids from cells, a condition that was employed to hydorlyze sialic acids in a number of literature reports 22,24,28,29 and therefore was not further optimized. This was followed by lyophilization and reaction with excessive DMB at 50 °C for 3 h. 29 After the reaction mixtures were filtered off through a 0.22 µm film, they were subjected to analysis by RP-HPLC using gradient mixtures of water and acetonitrile containing 0.1% TFA as eluents.…”

Section: Resultsmentioning

confidence: 99%

Quantifying the Efficiency ofN-Phenyl-D-mannosamine to Metabolically Engineer Sialic Acid on Cancer Cell Surface

Zhou¹,

Liao²,

Stepanovs³

et al. 2014

Journal of Carbohydrate Chemistry

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A convenient method was developed for the quantification of sialic acids expressed by cells and used to analyze the efficiency of N-phenylacetyl-D-mannosamine (ManNPhAc) to metabolically glycoengineer SKMEL-28 cancer cell. For this purpose, ManNPhAc-cultured cells were treated with 2M acetic acid to release sialic acids, and the products were treated with 1,2-diamino-4,5-methylenedioxybenzene to form the corresponding derivatives that had strong UV absorptions. The reaction mixture was then applied to HPLC-UV analysis to determine the amounts and the ratios of natural sialic acid and its unnatural analog. It was confirmed that after incubation with ManNPhAc SKMEL-28 cell was effectively glycoengineered to express a significant amount of unnatural sialic acid.

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“…2 Metabolic glycan labelling has been a powerful tool for labelling cell surface glycans in vitro and in vivo. [7][8][9][10][11] In this method, cells are fed with a non-natural monosaccharide carrying a small, inert chemical reporter group. This monosaccharide is then internalised by the cells and accepted by the biosynthetic machinery of glycoprotein synthesis in the Golgi and Endoplasmic Reticulum (ER).…”

Section: Introductionmentioning

confidence: 99%

Dual-sugar imaging using isonitrile and azido-based click chemistries

et al. 2013

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Imaging the Sialome during Zebrafish Development with Copper‐Free Click Chemistry

Cited by 85 publications

References 31 publications

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

Quantifying the Efficiency ofN-Phenyl-D-mannosamine to Metabolically Engineer Sialic Acid on Cancer Cell Surface

Dual-sugar imaging using isonitrile and azido-based click chemistries

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