Publications by authors named "Pauline M Rudd"

271 Publications

5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells.

Clin Epigenetics 2021 Feb 12;13(1):34. Epub 2021 Feb 12.

GlycoScience Group, the National Institute for Bioprocessing, Research and Training (NIBRT), Fosters Avenue, Mount Merrion, Blackrock, Co Dublin, Ireland.

Background: Glycosylation, one of the most fundamental post-translational modifications, is altered in cancer and is subject in part, to epigenetic regulation. As there are many epigenetic-targeted therapies currently in clinical trials for the treatment of a variety of cancers, it is important to understand the impact epi-therapeutics have on glycosylation.

Results: Ovarian and triple negative breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Branching and sialylation were increased on secreted N-glycans from chemo-sensitive/non-metastatic cell lines following treatment with 5-AZA-dC. These changes correlated with increased mRNA expression levels in MGAT5 and ST3GAL4 transcripts in ovarian cancer cell lines. Using siRNA transient knock down of GATA2 and GATA3 transcription factors, we show that these regulate the glycosyltransferases ST3GAL4 and MGAT5, respectively. Moreover, 5-AZA-dC-treated cells displayed an increase in migration, with a greater effect seen in chemo-sensitive cell lines. Western blots showed an increase in apoptotic and senescence (p21) markers in all 5-AZA-dC-treated cells. The alterations seen in N-glycans from secreted glycoproteins in 5-AZA-dC-treated breast and ovarian cancer cells were similar to the N-glycans previously known to potentiate tumour cell survival.

Conclusions: While the FDA has approved epi-therapeutics for some cancer treatments, their global effect is still not fully understood. This study gives insight into the effects that epigenetic alterations have on cancer cell glycosylation, and how this potentially impacts on the overall fate of those cells.
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http://dx.doi.org/10.1186/s13148-021-01015-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881483PMC
February 2021

N-Linked glycosylation profiles of therapeutic induced senescent (TIS) triple negative breast cancer cells (TNBC) and their extracellular vesicle (EV) progeny.

Mol Omics 2021 02 16;17(1):72-85. Epub 2020 Dec 16.

UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Dublin, Ireland.

Triple negative breast cancer (TNBC) has poor clinical outcomes and limited treatment options. Chemotherapy, while killing some cancer cells, can result in therapeutic-induced-senescent (TIS) cells. Senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Recently, N- and O-linked glycosylation alterations have been associated with senescence. We aimed to profile the N-linked glycans of whole cells, membrane, cytoplasm and EVs harvested from TIS TNBC cells and to compare these to results from non-senescent cells. TIS was induced in the Cal51 TNBC cells using the chemotherapeutic agent paclitaxel (PTX). Ultra-performance liquid chromatography (UPLC) analysis of exoglycosidase digested N-linked glycans was carried out on TIS compared to non-treated control cells. LC-Mass spectrometry (MS) analysis of the N-linked glycans and lectin blotting of samples was carried out to confirm the UPLC results. Significant differences were found in the N-glycan profile of the Cal51 membrane, cytoplasm and EV progeny of TIS compared to non-senescent cells. Protein mass spectrometry showed that the TIS cells contain different glycan modifying enzymes. The lectin, calnexin demonstrated a lower kDa size (∼58 kDa) in TIS compared to control cells (∼90 kDa) while Galectin 3 demonstrated potential proteolytic cleavage with 32 kDa and ∼22 kDa bands evident in TIS compared to non-senescent control cells with a major 32 kDa band only. TIS CAL51 cells also demonstrated a reduced adhesion to collagen I compared to control non-senescent cells. This study has shown that therapeutic-induced-senescent TNBC cells and their EV progeny, display differential N-glycan moieties compared to non-senescent Cal51 cells and their resultant EV progeny. For the future, N-glycan moieties on cancer senescent cells and their EV progeny hold potential for (i) the monitoring of treatment response as a liquid biopsy, and (ii) cancer senescent cell targeting with lectin therapies.
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http://dx.doi.org/10.1039/d0mo00017eDOI Listing
February 2021

Glycosylation in Indolent, Significant and Aggressive Prostate Cancer by Automated High-Throughput -Glycan Profiling.

Int J Mol Sci 2020 Dec 3;21(23). Epub 2020 Dec 3.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, A94 X099 Co. Dublin, Ireland.

The diagnosis and treatment of prostate cancer (PCa) is a major health-care concern worldwide. This cancer can manifest itself in many distinct forms and the transition from clinically indolent PCa to the more invasive aggressive form remains poorly understood. It is now universally accepted that glycan expression patterns change with the cellular modifications that accompany the onset of tumorigenesis. The aim of this study was to investigate if differential glycosylation patterns could distinguish between indolent, significant, and aggressive PCa. Whole serum -glycan profiling was carried out on 117 prostate cancer patients' serum using our automated, high-throughput analysis platform for glycan-profiling which utilizes ultra-performance liquid chromatography (UPLC) to obtain high resolution separation of -linked glycans released from the serum glycoproteins. We observed increases in hybrid, oligomannose, and biantennary digalactosylated monosialylated glycans (M5A1G1S1, M8, and A2G2S1), bisecting glycans (A2B, A2(6)BG1) and monoantennary glycans (A1), and decreases in triantennary trigalactosylated trisialylated glycans with and without core fucose (A3G3S3 and FA3G3S3) with PCa progression from indolent through significant and aggressive disease. These changes give us an insight into the disease pathogenesis and identify potential biomarkers for monitoring the PCa progression, however these need further confirmation studies.
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http://dx.doi.org/10.3390/ijms21239233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730228PMC
December 2020

Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow.

Anal Chem 2020 12 9;92(23):15323-15335. Epub 2020 Nov 9.

Bioprocessing Technology Institute, Agency for Science Technology and Research, 20 Biopolis Way, Biopolis 138668, Singapore.

High-throughput glycan analysis has become an important part of biopharmaceutical production and quality control. However, it is still a significant challenge in the field of glycomics to easily deduce isomeric glycan structures, especially in a high-throughput manner. Ion mobility spectrometry (IMS) is an excellent tool for differentiating isomeric glycan structures. However, demonstrations of the utility of IMS in high-throughput workflows such as liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) workflows have been limited with only a small amount of collision cross section (CCS) data available. In particular, IMS data of glycan fragments obtained in positive ion mode are limited in comparison to those obtained in negative ion mode despite positive ion mode being widely used for glycomics. Here, we describe IMS CCSN data obtained from a high-throughput LC-FLR-IMS-MS workflow in positive ion mode. We obtained IMS data from a selection of RapiFluor-MS (RFMS) labeled -glycans and also glycopeptides. We describe how IMS is able to distinguish isomeric -glycans and glycopeptides using both intact IMS and fragment-based IMS glycan sequencing experiments in positive ion mode, without significantly altering the high-throughput nature of the analysis. For the first time, we were able to successfully use IMS in positive ion mode to determine the branching of isomeric glycopeptides and RFMS labeled glycans. Further, we highlight that IMS glycan sequencing of fragments obtained from RFMS labeled glycans was similar to that of glycopeptides. Finally, we show that the IMS glycan sequencing approach can highlight shared structural features of nonisomeric glycans in a high-throughput LC-FLR-IMS-MS workflow.
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http://dx.doi.org/10.1021/acs.analchem.0c01954DOI Listing
December 2020

Clustering and curation of electropherograms: an efficient method for analyzing large cohorts of capillary electrophoresis glycomic profiles for bioprocessing operations.

Beilstein J Org Chem 2020 27;16:2087-2099. Epub 2020 Aug 27.

Analytics Group, Bioprocessing Technology Institute - Agency for Science Technology and Research. Singapore 138668.

The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughput generation of large amounts of glycomics data. This allows bioprocess engineers to identify critical process parameters that control the glycosylation critical quality attributes. The advances made in protocols for capillary electrophoresis-laser-induced fluorescence (CE-LIF) measurements of antibody N-glycans have increased the potential for generating large datasets of N-glycosylation values for assessment. With large cohorts of CE-LIF data, peak picking and peak area calculations still remain a problem for fast and accurate quantitation, despite the presence of internal and external standards to reduce misalignment for the qualitative analysis. The peak picking and area calculation problems are often due to fluctuations introduced by varying process conditions resulting in heterogeneous peak shapes. Additionally, peaks with co-eluting glycans can produce peaks of a non-Gaussian nature in some process conditions and not in others. Here, we describe an approach to quantitatively and qualitatively curate large cohort CE-LIF glycomics data. For glycan identification, a previously reported method based on internal triple standards is used. For determining the glycan relative quantities our method uses a clustering algorithm to 'divide and conquer' highly heterogeneous electropherograms into similar groups, making it easier to define peaks manually. Open-source software is then used to determine peak areas of the manually defined peaks. We successfully applied this semi-automated method to a dataset (containing 391 glycoprofiles) of monoclonal antibody biosimilars from a bioreactor optimization study. The key advantage of this computational approach is that all runs can be analyzed simultaneously with high accuracy in glycan identification and quantitation and there is no theoretical limit to the scale of this method.
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http://dx.doi.org/10.3762/bjoc.16.176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476600PMC
August 2020

Hypoxia Alters Epigenetic and -Glycosylation Profiles of Ovarian and Breast Cancer Cell Lines .

Front Oncol 2020 29;10:1218. Epub 2020 Jul 29.

GlycoScience Group, The National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland.

Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Our objective was to investigate these differential parameters, in an model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O) and compared to normoxia (21% O). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted -glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia ( ≤ 0.05). SiRNA transient knock down of transcription factors resulted in a decrease in the expression of glycosyltransferases and , which are responsible for sialylation and branching, respectively. These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells .
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http://dx.doi.org/10.3389/fonc.2020.01218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405916PMC
July 2020

Region-Specific Characterization of -Glycans in the Striatum and Substantia Nigra of an Adult Rodent Brain.

Anal Chem 2020 10 14;92(19):12842-12851. Epub 2020 Sep 14.

GlycoScience Group, National Institute for Bioprocessing Research and Training (NIBRT), Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin A94X099, Ireland.

-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of -glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of -glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson's disease. Specific objectives of the study include isolation of -glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of -glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total -glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type -glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN -glycans with the detection of phosphorylated -glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of -glycans in the striatum and SN of rodent brains, serving as a foundation for identifying "brain-type" glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders.
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http://dx.doi.org/10.1021/acs.analchem.0c01206DOI Listing
October 2020

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Sci Rep 2020 01 30;10(1):1464. Epub 2020 Jan 30.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.
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http://dx.doi.org/10.1038/s41598-019-57393-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992666PMC
January 2020

IgG Fc glycosylation as an axis of humoral immunity in childhood.

J Allergy Clin Immunol 2020 02 24;145(2):710-713.e9. Epub 2019 Oct 24.

Division of Immunology, Boston Children's Hospital, Boston, Mass; Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Boston, Mass. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010538PMC
February 2020

Sialic acid mediated mechanical activation of β adrenergic receptors by bacterial pili.

Nat Commun 2019 10 18;10(1):4752. Epub 2019 Oct 18.

Inserm, U1016, CNRS UMR8104, Institut Cochin, Université de Paris, Paris, France.

Meningococcus utilizes β-arrestin selective activation of endothelial cell β adrenergic receptor (βAR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that βAR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on βAR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host.
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http://dx.doi.org/10.1038/s41467-019-12685-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800425PMC
October 2019

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Mol Cell Proteomics 2020 01 7;19(1):11-30. Epub 2019 Oct 7.

Graduate School of Analytical Science and Technology, Chungnam National University, Gung-dong 220, Yuseong-Gu, Daejeon 305-764, Korea (South).

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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http://dx.doi.org/10.1074/mcp.RA119.001677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944243PMC
January 2020

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Biochemistry 2020 09 11;59(34):3123-3128. Epub 2019 Oct 11.

Bioprocessing Technology Institute, Agency for Science Technology and Research, Singapore 138668.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.
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http://dx.doi.org/10.1021/acs.biochem.9b00563DOI Listing
September 2020

A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study.

Mol Cell Proteomics 2019 11 30;18(11):2191-2206. Epub 2019 Aug 30.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland, A94X099.

The direct association of the genome, transcriptome, metabolome, lipidome and proteome with the serum glycome has revealed systems of interconnected cellular pathways. The exact roles of individual glycoproteomes in the context of disease have yet to be elucidated. In a move toward personalized medicine, it is now becoming critical to understand disease pathogenesis, and the traits, stages, phenotypes and molecular features that accompany it, as the disruption of a whole system. To this end, we have developed an innovative technology on an automated platform, "GlycoSeqCap," which combines -glycosylation data from six glycoproteins using a single source of human serum. Specifically, we multiplexed and optimized a successive serial capture and glycoanalysis of six purified glycoproteins, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), transferrin (Trf), haptoglobin (Hpt) and alpha-1-antitrypsin (A1AT), from 50 μl of human serum. We provide the most comprehensive and in-depth glycan analysis of individual glycoproteins in a single source of human serum to date. To demonstrate the technological application in the context of a disease model, we performed a pilot study in an ovarian cancer cohort ( = 34) using discrimination and classification analyses to identify aberrant glycosylation. In our sample cohort, we exhibit improved selectivity and specificity over the currently used biomarker for ovarian cancer, CA125, for early stage ovarian cancer. This technology will establish a new state-of-the-art strategy for the characterization of individual serum glycoproteomes as a diagnostic and monitoring tool which represents a major step toward understanding the changes that take place during disease.
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http://dx.doi.org/10.1074/mcp.RA119.001531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823853PMC
November 2019

Towards a standardized bioinformatics infrastructure for N- and O-glycomics.

Nat Commun 2019 07 22;10(1):3275. Epub 2019 Jul 22.

Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, 40530, Sweden.

The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.
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http://dx.doi.org/10.1038/s41467-019-11131-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796180PMC
July 2019

Combining Glucose Units, /, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer.

Anal Chem 2019 07 27;91(14):9078-9085. Epub 2019 Jun 27.

Analytics Group, Bioprocessing Technology Institute , Agency for Science, Technology and Research (A*STAR) , Singapore 138668.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.
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http://dx.doi.org/10.1021/acs.analchem.9b01476DOI Listing
July 2019

GlycopeptideGraphMS: Improved Glycopeptide Detection and Identification by Exploiting Graph Theoretical Patterns in Mass and Retention Time.

Anal Chem 2019 06 23;91(11):7236-7244. Epub 2019 May 23.

Bioprocessing Technology Institute , 20 Biopolis Way #06-01 , Singapore 138668.

The leading proteomic method for identifying N-glycosylated peptides is liquid chromatography coupled with tandem fragmentation mass spectrometry (LCMS/MS) followed by spectral matching of MS/MS fragment masses to a database of possible glycan and peptide combinations. Such database-dependent approaches come with challenges such as needing high-quality informative MS/MS spectra, ignoring unexpected glycan or peptide sequences, and making incorrect assignments because some glycan combinations are equivalent in mass to amino acids. To address these challenges, we present GlycopeptideGraphMS, a graph theoretical bioinformatic approach complementary to the database-dependent method. Using the AXL receptor tyrosine kinase (AXL) as a model glycoprotein with multiple N-glycosylation sites, we show that those LCMS features that could be grouped into graph networks on the basis of glycan mass and retention time differences were actually N-glycopeptides with the same peptide backbone but different N-glycan compositions. Conversely, unglycosylated peptides did not exhibit this grouping behavior. Furthermore, MS/MS sequencing of the glycan and peptide composition of just one N-glycopeptide in the graph was sufficient to identify the rest of the N-glycopeptides in the graph. By validating the identifications with exoglycosidase cocktails and MS/MS fragmentation, we determined the experimental false discovery rate of identifications to be 2.21%. GlycopeptideGraphMS detected more than 500 unique N-glycopeptides from AXL, triple the number found by a database search with Byonic software, and detected incorrect assignments due to a nonspecific protease cleavage. This method overcomes some limitations of the database approach and is a step closer to comprehensive automated glycoproteomics.
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http://dx.doi.org/10.1021/acs.analchem.9b00594DOI Listing
June 2019

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

Circ Genom Precis Med 2019 04 7;12(4):e002433. Epub 2019 Mar 7.

University of California San Diego School of Medicine (J.O., C.S., S.H., A.M.K., L.B.D., A.H.T., J.C.B.).

Background: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients.

Methods: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers.

Results: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood.

Conclusions: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.
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http://dx.doi.org/10.1161/CIRCGEN.118.002433DOI Listing
April 2019

Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity.

J Immunol 2019 04 22;202(8):2240-2253. Epub 2019 Feb 22.

Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland.

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele ( ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0-AAT bound IL-8 and leukotriene B, comparable to healthy control M-AAT, and significantly decreased leukotriene B-induced neutrophil adhesion ( = 0.04). Through a mechanism involving increased mRNA stability ( = 0.007), ataluren treatment of HEK-293 significantly increased mRNA expression ( = 0.03) and Q0-AAT truncated protein secretion ( = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.
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http://dx.doi.org/10.4049/jimmunol.1801045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452030PMC
April 2019

The minimum information required for a glycomics experiment (MIRAGE) project: LC guidelines.

Glycobiology 2019 05;29(5):349-354

Beilstein-Institut, Trakehner Str. 7-9, Frankfurt am Main, Germany.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to design guidelines that improve the reporting and reproducibility of glycoanalytical methods. Previously, the MIRAGE Commission has published guidelines for describing sample preparation methods and the reporting of glycan array and mass spectrometry techniques and data collections. Here, we present the first version of guidelines that aim to improve the quality of the reporting of liquid chromatography (LC) glycan data in the scientific literature. These guidelines cover all aspects of instrument setup and modality of data handling and manipulation and is cross-linked with other MIRAGE recommendations. The most recent version of the MIRAGE-LC guidelines is freely available at the MIRAGE project website doi:10.3762/mirage.4.
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http://dx.doi.org/10.1093/glycob/cwz009DOI Listing
May 2019

Clinical, neuroradiological, and biochemical features of SLC35A2-CDG patients.

J Inherit Metab Dis 2019 05 11;42(3):553-564. Epub 2019 Feb 11.

Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia.

SLC35A2-CDG is caused by mutations in the X-linked SLC35A2 gene encoding the UDP-galactose transporter. SLC35A2 mutations lead to hypogalactosylation of N-glycans. SLC35A2-CDG is characterized by severe neurological symptoms and, in many patients, early-onset epileptic encephalopathy. In view of the diagnostic challenges, we studied the clinical, neuroradiological, and biochemical features of 15 patients (11 females and 4 males) with SLC35A2-CDG from various centers. We describe nine novel pathogenic variations in SLC35A2. All affected individuals presented with a global developmental delay, and hypotonia, while 70% were nonambulatory. Epilepsy was present in 80% of the patients, and in EEG hypsarrhythmia and findings consistent with epileptic encephalopathy were frequently seen. The most common brain MRI abnormality was cerebral atrophy with delayed myelination and multifocal inhomogeneous abnormal patchy white matter hyperintensities, which seemed to be nonprogressive. Thin corpus callosum was also common, and all the patients had a corpus callosum shorter than normal for their age. Variable dysmorphic features and growth deficiency were noted. Biochemically, normal mucin type O-glycosylation and lipid glycosylation were found, while transferrin mass spectrometry was found to be more specific in the identification of SLC35A2-CDG, as compared to routine screening tests. Although normal glycosylation studies together with clinical variability and genetic results complicate the diagnosis of SLC35A2-CDG, our data indicate that the combination of these three elements can support the pathogenicity of mutations in SLC35A2.
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http://dx.doi.org/10.1002/jimd.12055DOI Listing
May 2019

High-throughput Serum -Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

Mol Cell Proteomics 2019 01 21;18(1):3-15. Epub 2018 Sep 21.

From the ‡Center for Proteomics and Metabolomics.

-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum -glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released -glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein -glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein -glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity -glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity -glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein -glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.
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http://dx.doi.org/10.1074/mcp.RA117.000454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317482PMC
January 2019

GlycanAnalyzer: software for automated interpretation of N-glycan profiles after exoglycosidase digestions.

Bioinformatics 2019 02;35(4):688-690

Bioprocessing Technology Institute Agency for Science, Technology and Research (A*STAR), Singapore.

Summary: Many eukaryotic proteins are modified by N-glycans. Liquid chromatography (ultra-performance -UPLC and high-performance-HPLC) coupled with mass spectrometry (MS) is conventionally used to characterize N-glycan structures. Software can automatically assign glycan structures by matching their observed retention times and masses with standardized values in reference databases. However, more precise confirmation of N-glycan structures can be derived using exoglycosidases, enzymes that remove specific monosaccharides from glycans. Exoglycosidase removal of monosaccharides results in signature peak shifts, in both UPLC and MS1, yielding an effective way to verify N-glycan structure with high detail (down to the position and isomeric linkage of each monosaccharide). Because manual interpretation of exoglycosidase data is complex and time consuming, we developed GlycanAnalyzer, a web application that pattern matches N-glycan peak shifts following exoglycosidase digestion and automates structure assignments. GlycanAnalyzer significantly improves assignment accuracy over other auto-assignment methods on tests with a monoclonal antibody and four glycan standards (100% versus 82% for the next best software). By automating data interpretation, GlycanAnalyzer enables the easier use of exoglycosidases to precisely define N-glycan structure.

Availability And Implementation: http://glycananalyzer.neb.com. Datasets available online.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378934PMC
February 2019

A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

Sci Rep 2018 06 22;8(1):9504. Epub 2018 Jun 22.

New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA.

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.
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http://dx.doi.org/10.1038/s41598-018-27797-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015026PMC
June 2018

Integrating biomarkers across omic platforms: an approach to improve stratification of patients with indolent and aggressive prostate cancer.

Mol Oncol 2018 09 7;12(9):1513-1525. Epub 2018 Aug 7.

UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.

Classifying indolent prostate cancer represents a significant clinical challenge. We investigated whether integrating data from different omic platforms could identify a biomarker panel with improved performance compared to individual platforms alone. DNA methylation, transcripts, protein and glycosylation biomarkers were assessed in a single cohort of patients treated by radical prostatectomy. Novel multiblock statistical data integration approaches were used to deal with missing data and modelled via stepwise multinomial logistic regression, or LASSO. After applying leave-one-out cross-validation to each model, the probabilistic predictions of disease type for each individual panel were aggregated to improve prediction accuracy using all available information for a given patient. Through assessment of three performance parameters of area under the curve (AUC) values, calibration and decision curve analysis, the study identified an integrated biomarker panel which predicts disease type with a high level of accuracy, with Multi AUC value of 0.91 (0.89, 0.94) and Ordinal C-Index (ORC) value of 0.94 (0.91, 0.96), which was significantly improved compared to the values for the clinical panel alone of 0.67 (0.62, 0.72) Multi AUC and 0.72 (0.67, 0.78) ORC. Biomarker integration across different omic platforms significantly improves prediction accuracy. We provide a novel multiplatform approach for the analysis, determination and performance assessment of novel panels which can be applied to other diseases. With further refinement and validation, this panel could form a tool to help inform appropriate treatment strategies impacting on patient outcome in early stage prostate cancer.
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http://dx.doi.org/10.1002/1878-0261.12348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120220PMC
September 2018

GlycoStore: a database of retention properties for glycan analysis.

Bioinformatics 2018 09;34(18):3231-3232

Institute for Glycomics, Griffith University, Gold Coast, Australia.

Summary: GlycoStore is a curated chromatographic, electrophoretic and mass-spectrometry composition database of N-, O-, glycosphingolipid (GSL) glycans and free oligosaccharides associated with a range of glycoproteins, glycolipids and biotherapeutics. The database is built on publicly available experimental datasets from GlycoBase developed in the Oxford Glycobiology Institute and then the National Institute for Bioprocessing Research and Training (NIBRT). It has now been extended to include recently published and in-house data collections from the Bioprocessing Technology Institute (BTI) A*STAR, Macquarie University and Ludger Ltd. GlycoStore provides access to approximately 850 unique glycan structure entries supported by over 8500 retention positions determined by: (i) hydrophilic interaction chromatography (HILIC) ultra-high performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC with fluorescent detection; (ii) porous graphitized carbon (PGC) chromatography in combination with ESI-MS/MS detection; and (iii) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). GlycoStore enhances many features previously available in GlycoBase while addressing the limitations of the data collections and model of this popular resource. GlycoStore aims to support detailed glycan analysis by providing a resource that underpins current workflows. It will be regularly updated by expert annotation of published data and data obtained from the project partners.

Availability And Implementation: http://www.glycostore.org.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty319DOI Listing
September 2018

Plasma N-glycans in colorectal cancer risk.

Sci Rep 2018 06 5;8(1):8655. Epub 2018 Jun 5.

National Institute for Bioprocessing Research & Training, Dublin, Ireland.

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.
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http://dx.doi.org/10.1038/s41598-018-26805-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988698PMC
June 2018

N-glycan signatures identified in tumor interstitial fluid and serum of breast cancer patients: association with tumor biology and clinical outcome.

Mol Oncol 2018 06 14;12(6):972-990. Epub 2018 May 14.

Danish Cancer Society Research Center, Genome Integrity Unit, Breast Cancer Biology Group, Copenhagen, Denmark.

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer.
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http://dx.doi.org/10.1002/1878-0261.12312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983225PMC
June 2018

Validation of an automated ultraperformance liquid chromatography IgG N-glycan analytical method applicable to classical galactosaemia.

Ann Clin Biochem 2018 Sep 13;55(5):593-603. Epub 2018 Mar 13.

2 National Centre for Inherited Metabolic Diseases, The Mater Misericordiae University Hospital, Dublin, Ireland.

Background Classical galactosaemia (OMIM #230400) is a rare disorder of carbohydrate metabolism caused by deficiency of the galactose-1-phosphate uridyltransferase enzyme. The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems, remains poorly understood. Current clinical methods of biochemical monitoring lack precision and individualization with an identified need for improved biomarkers for this condition. Methods We report the development and detailed validation of an automated ultraperformance liquid chromatography N-glycan analytical method of high peak resolution applied to galactose incorporation into human serum IgG. Samples are prepared on 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labelling and post-labelling clean-up with solid-phase extraction. Results This method is shown to be accurate and precise with repeatability (cumulative coefficients of variation) of 2.0 and 8.5%, respectively, for G0/G1 and G0/G2 ratios. Both serum and processed N-glycan samples were found to be stable at room temperature and in freeze-thaw experiments. Conclusions This high-throughput method of IgG galactose incorporation is robust, affordable and simple. This method is validated with the potential to apply as a biomarker for treatment outcomes for galactosaemia.
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http://dx.doi.org/10.1177/0004563218762957DOI Listing
September 2018

Expression, Purification, and Biochemical Characterization of Human Afamin.

J Proteome Res 2018 03 20;17(3):1269-1277. Epub 2018 Feb 20.

Vitateq Biotechnology GmbH , A-6020 Innsbruck, Austria.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.
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http://dx.doi.org/10.1021/acs.jproteome.7b00867DOI Listing
March 2018