A systematic study of glycopeptide esterification for the semi‐quantitative determination of sialylation in antibodies

Oliveira, Andrey Giovanni Gomes de; Roy, Rini; Raymond, Céline; Bodnar, Edward; Tayi, Venkata S.; Butler, Michael; Durocher, Yves; Perreault, Hélène

doi:10.1002/rcm.7287

Cited by 23 publications

(19 citation statements)

References 32 publications

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“…As minor species, non-sialylated G0F, G1F, and G2F glycoforms are completely suppressed in these spectra. Control experiments [53] showed that the suppression of G0F, G1F, and G2F relative to G2FS and G2FS2 is not due to loss of non-sialylated species from the sample due to heat or other experimental procedures. As the negative charge of sialic acid is neutralized through esterification, the positive mode mass spectrometric sensitivity of containing one underivatized sialic acid, b G2FSiaNAz1 containing one SiaNAz, c G2FS2 containing two underivatized sialic acids, and d G2FS1SiaNAz1 containing one underivatized sialic acid and one derivatized SiaNAz G2FS and G2FS2 increases, and thus, their abundances vs G0F, G1F, and G2F also increase.…”

Section: Resultsmentioning

confidence: 97%

“…HPLC separation of glycopeptides Separation of glycopeptides was performed using a previously reported method [44,52] Esterification of glycopeptides This reaction was conducted according to a published method [53], adapted from conditions reported by Reiding et al for free glycans [54]. Briefly, 10-μg samples of tryptic mAb glycopeptides were reacted with HOBt and EDC, both 0.25 M in ethanol (10 μL).…”

Section: Introductionmentioning

confidence: 99%

“…c Same glycopeptides as a after esterification. The mass shift from a corresponds to (+56, −18 for the peptide backbone[53] combined with +41, +28 for SiaNAz)…”

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confidence: 99%

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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: 97%

Section: Introductionmentioning

confidence: 99%

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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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“…Furthermore, recent studies have explored the feasibility of measuring deprotonated glycopeptides in negative ion mode with (130) and without (131) derivatization. Innovative strategies for the derivatization of sialic acid residues have also been designed to achieve linkage-specific (␣2,3 versus ␣2,6-sialyl) information at the glycopeptide level (132) and to obtain more equal ionization response from neutral and sialylated glycopeptides (133). Furthermore, attempts to generate universal workflows using Pronase digestion and C18-PGC LC-MS/MS instead of the conventional RP-LC-MS/MS of tryptic glycopeptides were described (39,134).…”

Section: Ms Acquisition Strategies In Glycoproteomics-lc-ms/mentioning

confidence: 99%

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Thaysen‐Andersen

Packer

Schulz

2016

Molecular & Cellular Proteomics

177

196

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“…TKPREEQYNSTY and its fragments can be used for monitoring the progression of mAb digestion . Quantitation studies of EEQYNSTYR derivatives using MALDI–MS , HPLC–UV–MS , or esterification of the peptide followed by MALDI–MS/MS were carried out, although RP was employed.…”

Section: Analysis Of N‐ and O‐glycopeptidesmentioning

confidence: 99%

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

Ikegami

2019

J of Separation Science

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Applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i.e., glycosylated proteins represented by monoclonal antibodies are discussed in the manner of glycoproteomics. They can be analyzed using hydrophilic interaction chromatography in five different stages as (1) their intact forms, (2) their subunits, (3) N‐ and O‐glycopeptides digested by proteases, (4) N‐ and O‐glycans released from the glycoproteins or glycopeptides, and (5) monosaccharides. Hydrophilic interaction chromatography is a more useful tool in the order of (1) to (5). At the stages (4) and (5), quantitation of glycans and saccharides are also reported. Hydrophilic interaction chromatography is employed not only for analytical uses, but also pretreatment items as solid phase extraction, followed by reversed‐phase liquid chromatography separations. Comprehensive search results of these application of hydrophilic interaction chromatography are summarized in tables to show what kind of hydrophilic interaction chromatography columns are suitable for each step of analysis.Relationship of favored and less favored hydrophilic interaction chromatography columns and their separation characteristics such as hydrophilicity, and selectivity for structural difference, is also discussed. Analysis of the therapeutic peptides (not glycosylated) using hydrophilic interaction chromatography is summarized, too.

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A systematic study of glycopeptide esterification for the semi‐quantitative determination of sialylation in antibodies

Abstract: For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.

Cited by 23 publications

References 32 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

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

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