Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.Proteomic

Universal detection of foot and mouth disease virus based on the conserved VP0 protein

Background: Foot and mouth disease virus (FMDV), a member of the picornaviridae that causes vesicular disease in ungulates, has seven serotypes and a large number of strains, making universal detection challenging. The mature virion is made up of 4 structural proteins, virus protein (VP) 1 – VP4, VP1-VP3 of which form the outer surface of the particle and VP4 largely contained within. Prior to mature virion formation VP2 and VP4 occur together as VP0, a structural component of the pre-capsid which, as a result of containing the internal VP4 sequence, is relatively conserved among all strains and serotypes. Detection of VP0 might therefore represent a universal virus marker. Methods: FMDV virus protein 0 (VP0) was expressed in bacteria as a SUMO fusion protein and the SUMO carrier removed by site specific proteolysis. Rabbit polyvalent sera were generated to the isolated VP0 protein and their reactivity characterised by a number of immunoassays and by epitope mapping on peptide arrays. Results: The specific VP0 serum recognised a variety of FMDV serotypes, as virus and as virus-like-particles, by a variety of assay formats. Epitope mapping showed the predominant epitopes to occur within the unstructured but highly conserved region of the sequence shared among many serotypes. When immunogold stained VLPs were assessed by TEM analysis they revealed exposure of epitopes on the surface of some particles, consistent with particle breathing hitherto reported for some other picornaviruses but not for FMDV. Conclusion: A polyvalent serum based on the VP0 protein of FMDV represents a broadly reactive reagent capable of detection of many if not all FMDV isolates. The suggestion of particle breathing obtained with this serum suggests a reconsideration of the FMDV entry mechanism

Cholesterol and Lipoprotein Dynamics in a Hibernating Mammal

Hibernating mammals cease feeding during the winter and rely primarily on stored lipids to fuel alternating periods of torpor and arousal. How hibernators manage large fluxes of lipids and sterols over the annual hibernation cycle is poorly understood. The aim of this study was to investigate lipid and cholesterol transport and storage in ground squirrels studied in spring, summer, and several hibernation states. Cholesterol levels in total plasma, HDL and LDL particles were elevated in hibernators compared with spring or summer squirrels. Hibernation increased plasma apolipoprotein A-I expression and HDL particle size. Expression of cholesterol 7 alpha-hydroxylase was 13-fold lower in hibernators than in active season squirrels. Plasma triglycerides were reduced by fasting in spring but not summer squirrels. In hibernators plasma β-hydroxybutyrate was elevated during torpor whereas triglycerides were low relative to normothermic states. We conclude that the switch to a lipid-based metabolism during winter, coupled with reduced capacity to excrete cholesterol creates a closed system in which efficient use of lipoproteins is essential for survival

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

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 submit- Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia; 23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇ ic´ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany; 26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada; 27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore, Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363–883 Korea (South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363–700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon, Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158–8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey 08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20–22 rue Henri et Gilberte Goudier 63200 RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry, Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California 94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District, Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave, Davis, California 95616; 70Horva´ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary; 72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg, Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tu¨ bingen, Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007 Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 24, 2019, and in revised form, August 26, 2019 Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677 ER: NISTmAb Glycosylation Interlaboratory Study 12 Molecular & Cellular Proteomics 19.1 Downloaded from https://www.mcponline.org by guest on January 20, 2020 ted 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 communityderived 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. Molecular & Cellular Proteomics 19: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying > 110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.Proteomic