Mass Spectrometric Analysis of Mutant Mice

North, Simon J.; Jang-Lee, Jihye; Harrison, Rebecca; Canis, Kevin; Ismail, Mohd Nazri; Trollope, Alana; Antonopoulos, Aristotelis; Pang, Poh-Choo; Grassi, Paola; Al-Chalabi, Sara; Etienne, A. Tony; Dell, Anne; Haslam, Stuart M.

doi:10.1016/s0076-6879(10)78002-2

Cited by 52 publications

(46 citation statements)

References 73 publications

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“…N-glycomic analysis was performed according to a protocol described previously59. Briefly, 50 μg of each sample was reduced by dithiothreitol (Sigma, Aldrich) and then carboxymethylated by iodoacetic acid (Sigma Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

Czajkowsky

Andersen

Fuchs

et al. 2015

Sci Rep

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The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.

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Section: Methodsmentioning

confidence: 99%

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

Czajkowsky

Andersen

Fuchs

et al. 2015

Sci Rep

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“…Sample Preparation, MS Data Acquisition, and Manual Data Analysis-Sample processing for N-glycomic profiling of the mouse tissues was carried out as detailed previously (3,4). The lung tissue preparations were subjected to homogenization, reduction, carboxymethylation, and tryptic digestion.…”

Section: Methodsmentioning

confidence: 99%

“…GC/MS Linkage Analysis-GC/MS linkage analysis of partially methylated alditol acetates was carried out on a PerkinElmer Life Sciences Clarus 500 instrument fitted with an RTX-5 fused capillary column (Restec Corp.) as described elsewhere (3,4).…”

Section: Methodsmentioning

confidence: 99%

“…Glycosylation is one of the most common and important post-translational modifications of proteins, yet it is also one of the most difficult to study because of its great complexity. Much of what we know about glycosylation, especially tissueand organism-specific variation, has been gathered via mass spectrometric profiling of detached glycans (1)(2)(3)(4). International, multi-laboratory comparisons of MS 1 and chromatographic glycomic data derived from standardized glycoproteins have concluded that the MS analysis of permethylated derivatives of released N-and O-linked glycans is the most robust glycomics strategy with respect to both sensitivity and quantitative reliability (5,6).…”

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confidence: 99%

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Polylactosaminoglycan Glycomics: Enhancing the Detection of High-molecular-weight N-glycans in Matrix-assisted Laser Desorption Ionization Time-of-flight Profiles by Matched Filtering

Bern

Brito

Pang

et al. 2013

Molecular & Cellular Proteomics

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For over 30 years, protocols based on the mass spectrometry (MS) of permethylated derivatives, complemented by enzymatic degradations, have underpinned glycomic experiments aimed at defining the structures of individual glycans present in the complex mixtures that are characteristic of biological samples. Both MS instrumentation and sample handling have improved markedly in recent years, enabling greater sensitivity and better signal-to-noise ratios, thereby facilitating the detection of glycans at much higher masses than could be achieved in the past. The latter is especially important for the characterization of the biologically important class of N-glycans that carry polylactosaminoglycan chains. Such advances in data acquisition heighten the need for informatics tools to assist in glycan structure assignment. Here, utilizing mouse lung tissue as a model system, we present evidence of polylactosaminoglycan-containing N-glycans with permethylated molecular weights exceeding 13 kDa. We show that antennae branching patterns and lengths can be successfully determined at these high masses via MS/MS experiments, even when MS ion counts are very low. We also describe the development and application of a matched filtering algorithm for assisting highmolecular-weight glycan detection and structure assignment. Glycosylation is one of the most common and important post-translational modifications of proteins, yet it is also one of the most difficult to study because of its great complexity. Much of what we know about glycosylation, especially tissueand organism-specific variation, has been gathered via mass spectrometric profiling of detached glycans (1-4). International, multi-laboratory comparisons of MS 1 and chromatographic glycomic data derived from standardized glycoproteins have concluded that the MS analysis of permethylated derivatives of released N-and O-linked glycans is the most robust glycomics strategy with respect to both sensitivity and quantitative reliability (5, 6). MS/permethylation strategies were first implemented in the Imperial laboratory in the early 1980s, when fast atom bombardment MS was revolutionizing the analysis of glycopolymers (7). Notably, with the exception of MALDI superseding fast atom bombardment as the preferred method of MS ionization in the past decade, permethylation-based glycomic protocols remain virtually unchanged from those employed 30 years ago. Where there has been a dramatic change, however, is in the quality and quantity of data emerging from glycomics investigations. This is exemplified by the success of the Analytical Glycotechnology Core of the Consortium for Functional Glycomics, which has built a public database of MALDI-TOF profiles from human and mouse tissues and cell lines as a resource for researchers worldwide.Characterizing complex mixtures of most classes of Nand/or O-glycans is now a routine task for glycomics experts when working with a few micrograms of a glycoprotein sample or, for cell glycomics, about a million cells. However, this is not the case fo...

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“…Mass Spectrometry Analysis-Sample processing for N-and O-glycomic profiling of the mouse tissues was carried out as described previously (37,38). The tissue preparations were subjected to homogenization, reduction, carboxymethylation, and tryptic digestion.…”

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confidence: 99%

Systemic Blockade of Sialylation in Mice with a Global Inhibitor of Sialyltransferases

Macauley

Arlian

Rillahan

et al. 2014

Journal of Biological Chemistry

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Background:In vivo pharmacological inhibition of sialyltransferases has, to date, not been possible. Results: 3F-NeuAc acts as a global sialyltransferase inhibitor in mice and causes kidney and liver dysfunction. Conclusion: Sialoside expression can be modulated in vivo with a sialyltransferase inhibitor. Significance: Pharmacological blockade of sialoside expression will be an important tool for future exploration of sialic acid in health and disease.

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Mass Spectrometric Analysis of Mutant Mice

Cited by 52 publications

References 73 publications

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

Polylactosaminoglycan Glycomics: Enhancing the Detection of High-molecular-weight N-glycans in Matrix-assisted Laser Desorption Ionization Time-of-flight Profiles by Matched Filtering

Systemic Blockade of Sialylation in Mice with a Global Inhibitor of Sialyltransferases

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