Papers by Morten Thaysen-andersen
Analytical Chemistry, Feb 27, 2019
Deep characterisation of biologically-relevant glycans remains challenging. Porous graphitised ca... more Deep characterisation of biologically-relevant glycans remains challenging. Porous graphitised carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat Protoc (7)1299:2012). Specifically, the ion intensities and signal-to-noise ratios of various classes of non-derivatised glycans from Nand O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN-and IPA-based PCMF dramatically increased the signal response across all glycan types (30-100x), improved the MS/MS spectral quality and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early-eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) whilst neither compromising the favourable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behaviour. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage and quantitative accuracy not least for the difficult-todetect early-eluting and low-abundance glycans detached from Nand O-glycoproteins.
Chemical Communications, 2014
Glycobiology, Dec 1, 2016
"The deleterious effect of AB5 toxins on Campylobacter jejuni strains that mimic GM1 ganglioside:... more "The deleterious effect of AB5 toxins on Campylobacter jejuni strains that mimic GM1 ganglioside: a means of bacterial warfare.
Nature Reviews Methods Primers, Jun 23, 2022
Protein glycosylation refers to the covalent attachment of carbohydrates to polypeptides and repr... more Protein glycosylation refers to the covalent attachment of carbohydrates to polypeptides and represents a class of prevalent and structurally diverse co-translational and post-translational modifications (PTMs) that impact a huge number of biological processes 1-6. Carbohydrate modifications include single monosaccharides and complex carbohydrate chains, both referred to as glycans. Protein glycosylation is a non-templated process and is mediated by enzymes known as glycosyltransferases, responsible for the initiation or elongation of glycans, and oligosaccharyltransferases, responsible for the addition of whole carbohydrate chains. In cells, the complex interplay between glycosyltransferases or oligosaccharyltransferases, carbohydrate transporters and glycosidases-the enzymes that remove these carbohydrates-fine-tunes the glycan structures observed on individual proteins and regulates glycoprotein function, with effects on biological processes that include cellular development 7 , cell-cell communication 8 , hostmicroorganism interactions 9,10 and immunity 5,11,12. For example, the recruitment of leukocytes to sites of inflammation is precisely controlled by specific glycan structures that mediate interactions with cell-surface lectins to enable selective and site-specific leukocyte homing 5,7,11,12. Dysregulation of glycosylation is associated with numerous diseases, including cancer 13-16 , infection and inflammation 17-22 , schizophrenia 23 and a wide range of congenital and neurological disorders 24-26. Unravelling the role of glycosylation under both physiological and pathophysiological conditions is a long-standing goal of glycobiology and has driven the rapid development of methods to track glycosylation for diagnostic and therapeutic purposes 27,28. Glycosylation is a universal protein modification across all domains of life with structurally distinct subclasses and glycan types now recognized 29-34 (Fig. 1a,b). Our knowledge of mammalian asparagine-linked (N-linked) and serine/threonine-linked (O-linked) glycans is the most developed, and these modifications are therefore the focus of this Primer. Characterizing the glycoproteome involves the identification of glycoproteins as well as definition of the macroheterogeneity (structural diversity owing to the presence or absence of glycans at specific glycosylation sites) and microheterogeneity (structural diversity of glycosylation patterns at individual glycosylation sites) 35 within these proteins. Microheterogeneity can arise through differences in the number and type of individual monosaccharide residues within the glycan, the structural arrangements and branching patterns of these monosaccharides Non-templated A process that is not guided by a template, in contrast to templated processes such as DNA transcription and translation.
Biochemical Society Transactions, Jul 20, 2021
Glycobiology, Jun 17, 2015
Glycomics may assist in uncovering the structure-function relationships of protein glycosylation ... more Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography-tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N-glycans and an under-representation of complex N-glycans (P < 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated (P < 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N-glycome profiles. EGFR-specific N-glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P < 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N-glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.
Final Program: 59th ASMS Conference on Mass Spectrometry and Allied Topics, June 5-9, 2011, Denver, Colorado
Journal of the American Society for Mass Spectrometry, Mar 30, 2011
Endocrine connections, Jul 1, 2019
Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) usi... more Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.
Glycobiology, Oct 18, 2021
We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unu... more We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man 1-3 GlcNAc 2 Fuc 0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man 2-3 GlcNAc 2 Fuc 1 , relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative Nglycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB −/−) displayed dramatically reduced paucimannosylation relative
Molecular & Cellular Proteomics, Aug 1, 2017
bioRxiv (Cold Spring Harbor Laboratory), Jan 20, 2023
Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, ... more Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporal N-glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncated N-glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophil Nglycoproteome featuring an intriguing granule-specific N-glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation.
Emerging roles of protein mannosylation in inflammation and infection
Molecular Aspects of Medicine, 2016
Proteins are frequently modified by complex carbohydrates (glycans) that play central roles in ma... more Proteins are frequently modified by complex carbohydrates (glycans) that play central roles in maintaining the structural and functional integrity of cells and tissues in humans and lower organisms. Mannose forms an essential building block of protein glycosylation, and its functional involvement as components of larger and diverse α-mannosidic glycoepitopes in important intra- and intercellular glycoimmunological processes is gaining recognition. With a focus on the mannose-rich asparagine (N-linked) glycosylation type, this review summarises the increasing volume of literature covering human and non-human protein mannosylation, including their structures, biosynthesis and spatiotemporal expression. The review also covers their known interactions with specialised host and microbial mannose-recognising C-type lectin receptors (mrCLRs) and antibodies (mrAbs) during inflammation and pathogen infection. Advances in molecular mapping technologies have recently revealed novel immuno-centric mannose-terminating truncated N-glycans, termed paucimannosylation, on human proteins. The cellular presentation of α-mannosidic glycoepitopes on N-glycoproteins appears tightly regulated; α-mannose determinants are relative rare glycoepitopes in physiological extracellular environments, but may be actively secreted or leaked from cells to transmit potent signals when required. Simultaneously, our understanding of the molecular basis on the recognition of mannosidic epitopes by mrCLRs including DC-SIGN, mannose receptor, mannose binding lectin and mrAb is rapidly advancing, together with the functional implications of these interactions in facilitating an effective immune response during physiological and pathophysiological conditions. Ultimately, deciphering these complex mannose-based receptor-ligand interactions at the detailed molecular level will significantly advance our understanding of immunological disorders and infectious diseases, promoting the development of future therapeutics to improve patient clinical outcomes.
Molecular & cellular proteomics : MCP, Jan 4, 2015
Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental... more Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types and this has been associated with biological dysregulation. Here we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha treated adipocytes correlated with the regulation of ...
Glycobiology, Jan 17, 2015
Glycomics may assist in uncovering the structure-function relationships of protein glycosylation ... more Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-LC-MS/MS-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6±13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by Western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an over-representation of high mannose, hybrid and paucimannosidic type N-glycans and...
Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum
The Journal of biological chemistry, Jan 3, 2015
Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linke... more Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose1-3fucose0-1N-acetylglucosamine2Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidas...
Biochimica et biophysica acta, 2014
Site-specific structural characterization of glycoproteins is important for understanding the exa... more Site-specific structural characterization of glycoproteins is important for understanding the exact functional relevance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached glycans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and efficient sample preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC-MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more stre...
Molecular & cellular proteomics : MCP, 2007
A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a rec... more A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be hig...
Glycobiology, 2015
Although mucin O-glycosylation of sputum from individuals suffering from cystic fibrosis (CF) is ... more Although mucin O-glycosylation of sputum from individuals suffering from cystic fibrosis (CF) is known to be altered relative to their unaffected counterparts, protein N-glycosylation of CF sputum remains structurally and functionally under-characterized. We report the first N-glycome of soluble proteins in sputum derived from five CF patients, two pathogen-free and two pathogen-infected/colonized non-CF individuals suffering from other pulmonary conditions. N-Glycans were profiled using porous graphitized carbon-liquid chromatography-negative ion-tandem mass spectrometry following enzymatic release from sputum proteins. The composition, topology and linkage isomers of 68 N-glycans were characterized and relatively quantified. Recurring structural features in all sputum N-glycomes were terminal α2,6-sialylation, α1,6-core fucosylation, β1,4-bisecting GlcNAcylation and compositions indicating paucimannosylation. Despite covering different genotypes, age, gender and microbial flora, t...
N-glycosylation of the Reactive Centre Loop of Corticosteroid-Binding Globulin Regulate Neutrophil Elastase-Based Cleavage and Cortisol Release
Frontiers in Immunology, 2014
Glycoproteins perform extra-and intracellular functions in innate and adaptive immunity by lectin... more Glycoproteins perform extra-and intracellular functions in innate and adaptive immunity by lectin-based interactions to exposed glyco-determinants. Herein, we document and mechanistically explain the formation of subcellular-specific N-glycosylation determinants on glycoproteins trafficking through the shared biosynthetic machinery of human cells. LC-MS/MS-based quantitative glycomics showed that the secreted glycoproteins of eight human breast epithelial cells displaying diverse geno-and phenotypes consistently displayed more processed, primarily complex type, N-glycans than the highmannose-rich microsomal glycoproteins. Detailed subcellular glycome profiling of proteins derived from three breast cell lines (MCF7/MDA468/MCF10A) demonstrated that secreted glycoproteins displayed significantly more α-sialylation and α1,6-fucosylation, but less α-mannosylation, than both the intermediately glycan-processed cell-surface glycoproteomes and the under-processed microsomal glycoproteomes. Subcellular proteomics and gene ontology revealed substantial presence of endoplasmic reticulum resident glycoproteins in the microsomes and confirmed significant enrichment of secreted and cell-surface glycoproteins in the respective subcellular fractions.The solvent accessibility of the glycosylation sites on maturely folded proteins of the 100 most abundant putative N-glycoproteins observed uniquely in the three subcellular glycoproteomes correlated with the glycan type processing thereby mechanistically explaining the formation of subcellular-specific Nglycosylation. In conclusion, human cells have developed mechanisms to simultaneously and reproducibly generate subcellular-specific N-glycosylation using a shared biosynthetic machinery. This aspect of protein-specific glycosylation is important for structural and functional glycobiology and discussed here in the context of the spatio-temporal interaction of glyco-determinants with lectins central to infection and immunity.
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Papers by Morten Thaysen-andersen