Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases

Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase

Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmissiondeficits in long-term potentiationand specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients

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

Implication of the Kallikrein-Kinin System in Neurological Disorders: Quest for Potential Biomarkers and Mechanisms

Neurological disorders represent major health concerns in terms of comorbidity and mortality worldwide. Despite a tremendous increase in our understanding of the pathophysiological processes involved in disease progression and prevention, the accumulated knowledge so far resulted in relatively moderate translational benefits in terms of therapeutic interventions and enhanced clinical outcomes. Aiming at specific neural molecular pathways, different strategies have been geared to target the development and progression of such disorders. The kallikrein-kinin system (KKS) is among the most delineated candidate systems due to its ubiquitous roles mediating several of the pathophysiological features of these neurological disorders as well as being implicated in regulating various brain functions. Several experimental KKS models revealed that the inhibition or stimulation of the two receptors of the KKS system (B1R and B2R) can exhibit neuroprotective and/or adverse pathological outcomes. This updated review provides background details of the KKS components and their functions in different neurological disorders including temporal lobe epilepsy, traumatic brain injury, stroke, spinal cord injury, Alzheimer’s disease, multiple sclerosis and glioma. Finally, this work will highlight the putative roles of the KKS components as potential neurotherapeutic targets and provide future perspectives on the possibility of translating these findings into potential clinical biomarkers in neurological disease

Altered O-glycomes of Renal Brush-Border Membrane in Model Rats with Chronic Kidney Diseases

Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions

Recent updates on drug abuse analyzed by neuroproteomics studies: Cocaine, Methamphetamine and MDMA

Currently, drug abuse and addiction represent a global public health concern with about 13.6 million people using illicit drugs in the USA alone. Substance abuse intervenes in normal brain functioning, causing alterations in memory, behavior and neuronal physiology. Although many studies have been conducted to elucidate the mode of action of different drugs, the heterogeneous modes of drug intake led to a complicated profile of drug-induced brain changes involving neurotoxicity and addiction. Given the complex interplay of genes and proteins in mediating these effects, neuroproteomics analysis has been considered among the methods of choice to complement what has already been discovered and to create targeted therapies. In this review, we will focus on three drugs, namely cocaine, methamphetamine (METH) and 3,4-methylenedioxy-N-methylamphetamine (MDMA). In the context of neuroproteomics, these drugs have been extensively studied by utilizing different experimental models, including primate and non-primate animals along with postmortem human samples. Even though there are many variations in the results, these drugs were shown to employ common pathways in eliciting their effects. Neuroproteomics analysis of these drugs has led to the identification of differentially expressed proteins involved in metabolism, oxidative stress, cell signaling, cytoskeleton, cell death and synaptic plasticity. Finally, this work will discuss recent findings from our laboratory by looking at a model of chronic methamphetamine abuse and its effect on different brain regions

The Effect of Chronic Methamphetamine Exposure on the Hippocampal and Olfactory Bulb Neuroproteomes of Rats

<div><p>Nowadays, drug abuse and addiction are serious public health problems in the USA. Methamphetamine (METH) is one of the most abused drugs and is known to cause brain damage after repeated exposure. In this paper, we conducted a neuroproteomic study to evaluate METH-induced brain protein dynamics, following a two-week chronic regimen of an escalating dose of METH exposure. Proteins were extracted from rat brain hippocampal and olfactory bulb tissues and subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Both shotgun and targeted proteomic analysis were performed. Protein quantification was initially based on comparing the spectral counts between METH exposed animals and their control counterparts. Quantitative differences were further confirmed through multiple reaction monitoring (MRM) LC-MS/MS experiments. According to the quantitative results, the expression of 18 proteins (11 in the hippocampus and 7 in the olfactory bulb) underwent a significant alteration as a result of exposing rats to METH. 13 of these proteins were up-regulated after METH exposure while 5 were down-regulated. The altered proteins belonging to different structural and functional families were involved in processes such as cell death, inflammation, oxidation, and apoptosis.</p></div

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures

Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (<i>P</i> < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood–brain barrier during brain metastasis, were observed