Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography

Müthing, Johannes; Unland, Frank

doi:10.1016/0378-4347(94)00213-4

Cited by 40 publications

(11 citation statements)

References 20 publications

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“…LC has the additional advantages that it a ) reduces the complexity of the electrospray droplet, thereby decreasing the likelihood that it will be composed of compounds with greatly different gas phase basicity or acidity, which can suppress the ionization of the analyte(s) of interest ( 42 ); b ) separates salts in the biological extract from the compounds of interest; hence, it minimizes formation of multiple salt adducts and other sources of ionization suppression ( 43 ); c ) concentrates the analytes of interest in a small volume; and d ) with appropriate choice of LC conditions, separates isomers that might not be distinguished by MS or MS/MS alone ( 44,45 ).…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Shaner

Allegood

Park

et al. 2009

Journal of Lipid Research

384

366

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Section: Discussionmentioning

confidence: 99%

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Shaner

Allegood

Park

et al. 2009

Journal of Lipid Research

384

366

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“…Additionally, LC serves to concentrate the analytes of interest into a small elution volume thereby increasing sensitivity. Finally, LC may separate isomers (with appropriate choice of separation conditions) that might not be distinguished by MS or MS/MS alone [61, 62]. …”

Section: Discussionmentioning

confidence: 99%

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sullards

Liu

Chen

et al. 2011

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

126

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Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.

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“…Gangliosides were isolated from buttermilk according to standard procedures (Ledeen and Yu, 1982) and purified using TMAE-Fractogel chromatography (Müthing and Unland, 1994;Müthing, 2000). GM1 was obtained from Pallmann (Munich, Germany).…”

Section: Reference Gangliosidesmentioning

confidence: 99%

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti‐melanoma mouse IgG3 monoclonal antibody R24

Müthing

Kemminer

Conradt³

et al. 2003

Biotech & Bioengineering

Self Cite

100

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R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity.

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Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography

Cited by 40 publications

References 20 publications

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti‐melanoma mouse IgG3 monoclonal antibody R24

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