388 research outputs found
N-Glycan matrix-assisted laser desorption/ionization mass spectrometry imaging protocol for formalin-fixed paraffin-embedded tissues
No full textLink to a related website: https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/rcm.7845, Open Access via UnpaywallAbstract not availableMatthew T. Briggs, Yin Ying Ho, Gurjeet Kaur, Martin K. Oehler, Arun V. Everest-Dass, Nicolle H. Packer and Peter Hoffman
What comes next in glycobiology
No full textGlycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.Fil: Seeberger, Peter H.. Max Planck Institute Of Biochemistry.; AlemaniaFil: Ge, Yun. Shenzhen Bay Laboratory; ChinaFil: Szymanski, Christine M.. University of Georgia; Estados UnidosFil: Kolarich, Daniel. Griffith University. Griffith School Of Engineering; AustraliaFil: Thaysen Andersen, Morten. Nagoya University; JapónFil: Packer, Nicolle H.. Mcquarie University; AustraliaFil: Fadda, Elisa. University of Southampton; Reino UnidoFil: Davis, Benjamin. University of Oxford; Reino UnidoFil: Nishihara, Shoko. Soka University; JapónFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Kwong, Peter D.. National Institutes of Health; Estados Unidos. Columbia University; Estados UnidosFil: Strasser, Richard. University Of Natural Resources And Life Sciences
The molecular basis of immunosuppression by soluble CD52 is defined by interactions of N-linked and O-linked glycans with HMGB1 box B
No full textHuman soluble CD52 is a short glycopeptide comprising 12 amino acids (GQNDTSQTSSPS) which functions as an immune regulator by sequestering the pro-inflammatory high mobility group box protein 1 (HMGB1) and suppressing immune responses. Recombinant CD52 has been shown to act as a broad anti-inflammatory agent, dampening both adaptive and innate immune responses. This short glycopeptide is heavily glycosylated, with a complex sialylated N-linked glycan at N3 and reported O-linked glycosylation possible on several serine and threonine residues. Previously we demonstrated that specific glycosylation features of CD52 are essential for its immunosuppressive function, with terminal α-2,3-linked sialic acids required for binding to the inhibitory SIGLEC-10 receptor leading to T-cell suppression. Using high resolution mass spectrometry, we have further characterized the N- and O-linked glycosylation of Expi293 recombinantly produced CD52 at a glycopeptide and released glycan level, accurately determining glycan heterogeneity of both N- and O-linked glycosylation, and localizing the site of O-glycosylation to T8 with high confidence and direct spectral evidence. This detailed knowledge of CD52 glycosylation informed the construction of a model system, which we analyzed by molecular dynamics simulations to understand the mechanism of recognition and define interactions between bioactive CD52, HMGB1 and the SIGLEC-10 receptor. Our results confirm the essential role of glycosylation, more specifically hyper-sialylation, in the function of CD52, and identify at the atomistic level specific interactions between CD52 glycans and the Box B domain of HMGB1 that determine recognition, and the stability of the CD52/HMGB1 complex. These insights will inform the development of synthetic CD52 as an immunotherapeutic agent.Full Tex
MALDI mass spectrometry imaging of N-glycans on tibial cartilage and subchondral bone proteins in knee osteoarthritis
No full textAccepted manuscript online: 15 March 2016
Link to a related website: https://rss.onlinelibrary.wiley.com/doi/am-pdf/10.1002/pmic.201500461, Open Access via UnpaywallMagnetic Resonance Imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.Matthew T. Briggs, Julia S. Kuliwaba, Dzenita Muratovic, Arun V. Everest, Dass, Nicolle H. Packer, David M. Findlay, Peter Hoffman
Interaction between Polysialic Acid and the MARCKS-ED Peptide at the Molecular Level
No full textPolysialic acid (polySia) is a highly negatively charged linear homopolymer comprising α-2,8-linked sialic acids. It is abundant in the embryonic brain and modulates various functions such as differentiation and synaptic plasticity in the adult central nervous system by direct binding to its protein partners. One such example is the binding of polySia to myristoylated-alanine rich C-kinase substrate (MARCKS) to modulate neuritogenesis. To understand their interaction mechanism at the molecular level, we performed a binding assay which showed a direct binding of the MARCKS-ED peptide (KKKKKRFSFKKSFKLSGFSFKKNKK) with polySia in a concentration-dependent manner. Molecular dynamics simulations revealed that this binding is not exclusively dominated by electrostatics but can in part be attributed to the presence of near-regularly spaced Phe residues, that confer a compact 3D conformation based on pseudoglycine loop structures supported by Phe-Phe interactions. Our simulations, which are confirmed by circular dichroism measurements, also indicate that the peptide-polySia binding induces large-scale conformational rearrangement of polySia into coils at the binding site, whereas the peptide conformation is relatively unperturbed. As a consequence, we predict that each peptide can bind to a domain extending ∼14 polySia repeat units. Using the fluorescently tagged MARCKS-ED peptide on rat brainstem tissue sections, we demonstrate the ability of the peptide to detect polySia, similarly to polySia-specific antibody mAb735, especially in the spinal trigeminal nucleus and the dorsal vagal complex. This study provides information about the interaction between polySia and its CNS protein binding partner, MARCKS, and provides a fundamental platform for further studies to explore the prospect of the MARCKS-ED as an effective polySia-binding peptide for bioimaging and drug delivery applications.No Full Tex
MALDI mass spectrometry imaging of early- and late-stage serous ovarian cancer tissue reveals stage-specific N-glycans
No full textData source: Supporting information, https://doi.org/10.1002/pmic.201800482Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃ (GlcNAc)₂ , and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃ (GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.Matthew T. Briggs, Mark R. Condina, Yin Ying Ho, Arun V. Everest-Dass, Parul Mittal, Gurjeet Kaur, Martin K. Oehler, Nicolle H. Packer, and Peter Hoffman
MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney
No full textRecent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.Ove J. R. Gustafsson, Matthew T. Briggs, Mark R. Condina, Lyron J. Winderbaum, Matthias Pelzing, Shaun R. McColl, Arun V. Everest-Dass, Nicolle H. Packer, Peter Hoffman
Glycomics & Glycoproteomics: From Analytics to Function
No full textGlycomics and glycoproteomics define powerful technologies that help scientists to capture this biological “dark matter” and study all glycans and glycoproteins expressed by cells, tissues or organisms at a given time, space and condition.2–4 While both glycomics and glycoproteomics methods have existed for several years,5–8 key advances in the separation sciences, mass spectrometry and informatics have recently allowed for a more complete implementation and integration of glycomics and glycoproteomics in the life sciences. As exemplified by papers published in this themed issue in Molecular Omics “Glycomics & Glycoproteomics: From Analytics to Function”, studies that concertedly apply two or more glyco-centric ‘omics techniques to uncover new mechanisms in various research areas in glycobiology such as pathogen–host interactions (Cain et al., DOI: 10.1039/D0MO00032A; Mule et al., DOI: 10.1039/D0MO00043D; Delannoy et al., DOI: 10.1039/C9MO00173E; Mthembu et al., DOI: 10.1039/C9MO00175A) cancer and biomarkers (Chandler et al., DOI: 10.1039/D0MO00009D; Acharya et al., DOI: 10.1039/C9MO00061E) and embryogenesis (Qu et al., DOI: 10.1039/D0MO00005A), are becoming common in the literature. This themed issue also illustrates that mass spectrometry-based glycoproteomics studies often use multiple orthogonal fragmentation schemes to produce complementary glycan structural information to aid glycopeptide characterisation at scale, with sensitivity, speed and accuracy (Pap et al., DOI: 10.1039/C9MO00160C; Chalkley et al., DOI: 10.1039/C9MO00178F). Other new technology improvements (Cordina et al., DOI: 10.1039/C9MO00181F; Zhang et al., DOI: 10.1039/C9MO00180H; Sethi et al., DOI: 10.1039/D0MO00019A), informatics advancements (Chalkley et al., DOI: 10.1039/C9MO00178F; Uh et al., DOI: 10.1039/C9MO00174C; Phung et al., DOI: 10.1039/C9MO00125E) and pathway analyses (del Solar et al., DOI: 10.1039/D0MO00023J; Donald et al., DOI: 10.1039/C9MO00168A) also contribute to an ever-expanding analytical toolbox that now allows scientists to explore not only the structure but also the biosynthesis and function of glycans and glycoproteins.Full Tex
Rapid and sensitive glycan targeting by lectin-SERS assay
No full textGlycosylation is an important part of cell signalling that is implicated in many disease states in which glycans play an essential role. Therefore rapid and sensitive differentiation of glycans on proteins is highly desirable. Current technologies for glycan structural analysis normally involve the isolation of glycans from proteins, or enrichment of glycopeptides, and detection by mass spectrometry, which requires relatively large amounts of sample and is not able to be used by non-specialist laboratories. Herein we present a simple and new strategy for targeting the glycans on a protein (with IgG as a model glycoprotein) using surface-enhanced Raman scattering (SERS) coupled to glycan-binding WGA (wheat germ agglutinin) lectin, in a lectin-SERS assay. With one drop (1 μL) of glycoprotein solution, our lectin-SERS assay can detect as low as 10 ng IgG within two hours with high glycan specificity. We extend our technique to examine the surface glycan profiles on two human colorectal cancer cell lines, which show different and unique glycan signatures specific to the target cell lines. Thus, we believe that this method could be potentially used for the real-time and in situ monitoring of glycans on the surface of cells or tissue or in body fluids, and is thus a powerful tool for glycomics research.No Full Tex
Chemoenzymatic glycan labelling as a platform for site-specific IgM-antibody drug conjugates
No full textImmunoglobulin M (IgM) type antibodies play a significant role in complement activation, cellular debris clearance and cell quality control, and have the potential to be used as a therapeutic or targeting/delivery antibody. However, this potential has not been explored thoroughly due to its high molecular weight, polymeric structure and large number of glycosylation sites. Site-specific antibody-drug-conjugates (ADC) are considered the next generation protein biotherapeutic drugs and currently all, in clinical trials and approved, are of the IgG isotype. As existing methods for the development and characterization of IgG-ADCs are not compatible with IgM-ADC, we describe a platform methodology suitable for site specific IgM-ADC using a chemoenzymatic method targeting the glycans on the IgM. Azide functionalized sialic acids were incorporated onto IgM glycans using sialyltransferase for biocompatible conjugation using “click” chemistry. The number of azide groups incorporated onto the IgM glycans were characterized by mass spectrometry of the enzymatically released glycans and glycopeptides. Quantitation of the azide incorporation showed an azide antibody ratio of 8 (glycan data) and 6–10 (glycopeptide data) which translates to a high drug antibody ratio based on IgG-ADC standards. This platform methodology can be readily adapted for any human IgM produced in a mammalian cell expression system.No Full Tex
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