bims-glecem Biomed News
on Glycogen metabolism in exercise, cancer and energy metabolism
Issue of 2022–03–06
fiveteen papers selected by
Dipsikha Biswas, Københavns Universitet
  1. The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination.
  2. Formation and physicochemical properties of glycogen phosphorylase in complex with a cationic polyelectrolyte.
  3. A new phenotype of aldolase a deficiency in a 14 year-old boy with epilepsy and rhabdomyolysis - case report.
  4. AKT/GSK-3β signaling is altered through downregulation of mTOR during cerebral Ischemia/Reperfusion injury.
  5. Very early-onset inflammatory bowel disease: Novel description in glycogen storage disease type Ia.
  6. No effect of triheptanoin in patients with phosphofructokinase deficiency.
  7. 3.19 Inborn Errors of Metabolism.
  8. Mild disease course of SARS-CoV-2 infections and mild side effects of vaccination in Pompe disease: a cohort description.
  9. Clinical features of gout in adult patients with type Ia glycogen storage disease: a single-centre retrospective study and a review of literature.
  10. [Engineering the glycogen metabolism in cyanobacterial photosynthetic cell factories: a review].
  11. 73 Preimplantation genetic diagnosis of glycogen branching enzyme deficiency and sex determination in equine in vitro-produced embryos.
  12. Combined treatment with niclosamide ethanolamine and artemether combination improves type 1 diabetes via the targeting of liver mitochondria.
  13. Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.
  14. Urinary UDP-Glucose as a Novel Actionable Biomarker of Dehydration-Induced Acute Kidney Injury.
  15. In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.
  1. J Vis Exp. 2022 Feb 08.
    The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination.
    Ziyi Wang, Qinghua Liu, Liang Wang, Robert G Gilbert, Mitchell A Sullivan.
      Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.
    DOI:  https://doi.org/10.3791/63088
  2. Int J Biol Macromol. 2022 Feb 28. pii: S0141-8130(22)00388-9. [Epub ahead of print]
    Formation and physicochemical properties of glycogen phosphorylase in complex with a cationic polyelectrolyte.
    Dionysios D Neofytos, Aristeidis Papagiannopoulos, Evangelia D Chrysina, Stergios Pispas.
      The accumulation of rabbit muscle glycogen phosphorylase b (RMGPb) in electrostatic complexes with the cationic polyelectrolyte poly 2-(dimethylamino) ethyl methacrylate in its quenched form (QPDMAEMA) was studied in two buffer solutions. In the N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, large complexes of RMGPb-QPDMAEMA were formed which adopted smaller sizes as QPDMAEMA concentration increased. However, in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, the hydrodynamic radius of the formed complexes gradually increased as the polymer concentration increased. Zeta potential measurements (ζp) showed that RMGPb significantly changed the ζp of the QPDMAEMA aggregates. Fluorescence studies showed that the interaction between RMGPb and QPDMAEAMA was enhanced as polymer concentration increased. Specifically, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence indicated that in the BES buffer the aggregates became denser as more QPDMAEMA was added, while in the HEPES buffer the density of the formed structures decreased. RMGPb's secondary structure was examined by Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) and Circular Dichroism (CD) showing that QPDMAEMA interaction with RMGPb does not induce any changes to the secondary structure of the enzyme. These observations suggest that cationic polyelectrolytes may be utilized for the formulation of RMGPb in multifunctional nanostructures and be further exploited in innovative biotechnology applications and bioinspired materials development.
    Keywords:  Biophysical characterization; Cationic polyelectrolytes; Glycogen phosphorylase; Protein-polymer complexes
    DOI:  https://doi.org/10.1016/j.ijbiomac.2022.02.136
  3. Ital J Pediatr. 2022 Mar 04. 48(1): 39
    A new phenotype of aldolase a deficiency in a 14 year-old boy with epilepsy and rhabdomyolysis - case report.
    Lucia Santoro, Dorina Pjetraj, Virtut Velmishi, Carmen Campana, Carlo Catassi, Carlo Dionisi-Vici, Arianna Maiorana.
       BACKGROUND: Glycogen storage disease type XII is a rare metabolic disease resulting from Aldolase A deficiency that causes muscle glycogen accumulation, with crisis of rhabdomyolysis and hemolytic anemia. In the very few cases described, rhabdomyolysis crises are caused by fever and/or exercise and can accompany acute hemolytic anemia. Although currently there is no therapy available for this disease, the guidelines for the management of other forms of glycogen storage diseases recommend a nutritional therapy in order to avoid hypoglycemia or prevent exercise-induced rhabdomyolysis.
    CASE PRESENTATION: In this case report we describe a new phenotype of the disease in a 14-year-old boy, characterized by seizures and rhabdomyolysis. Beside an antiepileptic treatment, we propose a new therapeutic approach based on ketogenic diet in order to supply an energetic substrate for skeletal muscle and neurons.
    CONCLUSIONS: The anti-epileptic therapy and the dietetic approach were well tolerated by the patient who showed good compliance. This led to a deceleration of the disease with no other acute episodes of seizures and rhabdomyolysis, without any side effects observed.
    Keywords:  24 GSD XII; ALDOA Deficiency; Aldolase A Deficiency; Glycogen Storage Disease type XII; Ketogenic Diet
    DOI:  https://doi.org/10.1186/s13052-022-01228-3
  4. Mol Biol Rep. 2022 Mar 02.
    AKT/GSK-3β signaling is altered through downregulation of mTOR during cerebral Ischemia/Reperfusion injury.
    Shengwei Peng, Jin-Hua Gu, Chun-Ling Dai, Khalid Iqbal, Fei Liu, Cheng-Xin Gong.
       PURPOSE: Cellular responses following cerebral ischemia/reperfusion injury are critical to recovery and survival after ischemic stroke. Understanding of these cellular responses can help the design of therapies to protect brain tissue and promote recovery after stroke. One of these cellular responses may be mediated by the AKT (protein kinase B) signal transduction pathway. This study was aimed to investigate the cerebral ischemia-induced alterations of AKT signaling and the upstream molecular pathways.
    METHODS: We modeled cerebral ischemia by middle cerebral artery occlusion in 2-3-month-old male C57BL/6J mice and then analyze the brain samples by using quantitative Western blots and phosphorylation/activation-dependent kinase antibodies. Cerebral ischemia was confirmed by staining of brain slices with 1% 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl, as well as neurological assessments of the mice 24 h after ischemia-reperfusion surgery.
    RESULTS: We found marked downregulation of AKT within 12 h of cerebral ischemia/reperfusion, which leads to overactivation of glycogen synthase kinase-3β (GSK-3β). Furthermore, we found that the downregulation of AKT was mediated by downregulation of mTORC2 (the complex 2 of the mechanistic target of rapamycin) instead of its common upstream kinases, phosphatidylinositol 3-kinase and phosphoinositide-dependent kinase-1.
    CONCLUSION: Our findings provide new insight into the cellular responses to ischemia/reperfusion brain injury and will help develop new treatments targeting the AKT signaling pathway for the treatment of ischemic stroke.
    Keywords:  Cerebral ischemia/reperfusion; GSK-3β; Middle cerebral artery occlusion; Protein kinase B (AKT); Signal transduction; mTOR
    DOI:  https://doi.org/10.1007/s11033-022-07247-x
  5. Mol Genet Metab Rep. 2022 Jun;31 100848
    Very early-onset inflammatory bowel disease: Novel description in glycogen storage disease type Ia.
    William B Hannah, Ricardo C Ong, Margarita Nieto Moreno, Surekha Pendyal, Monica Abdelmalak, Judith Kelsen, Nancy M McGreal, Priya S Kishnani.
      Although inflammatory bowel disease is a well-described feature of glycogen storage disease type Ib, it has been reported in only a small number of individuals with glycogen storage disease type Ia (GSDIa). We describe, to our knowledge, the first patient with GSDIa and very early-onset inflammatory bowel disease (VEO-IBD). Larger studies are needed to better understand this possible association, elucidate the mechanism of VEO-IBD in GSDIa, and inform management.
    Keywords:  Glycogen storage disease type 1a; Very early-onset inflammatory bowel
    DOI:  https://doi.org/10.1016/j.ymgmr.2022.100848
  6. Neuromuscul Disord. 2022 Feb 04. pii: S0960-8966(22)00029-3. [Epub ahead of print]
    No effect of triheptanoin in patients with phosphofructokinase deficiency.
    Daniel Emil Raaschou-Pedersen, Karen Lindhardt Madsen, Nicoline Løkken, Jesper Helbo Storgaard, Ros Quinlivan, Pascal Laforêt, Allan Lund, Gerrit Van Hall, John Vissing, Mette Ørngreen.
      Phosphofructokinase deficiency (PFKD) is a rare disorder of glycogen metabolism. The lack of phosphofructokinase activity blocks the oxidative pathway from glucose and glycogen to pyruvate. Patients suffer from myopathy, exercise intolerance, and myoglobinuria. Currently, there is no specific treatment for PFKD. We hypothesized that 2 weeks treatment with triheptanoin could improve oxidative metabolism during exercise by bypassing the blocked pyruvate generation in PFKD. The study was a randomized, double-blind, placebo-controlled crossover study. Three genetically verified patients completed two treatment periods of 14 days each with triheptanoin (0.3-1 g × kg-1 × day-1) or placebo liquid. Primary outcomes were heart rate, fatty acid and total oxidation measured via stable isotope and indirect calorimetry methodology during submaximal exercise. Triheptanoin did not improve the primary outcome heart rate during submaximal exercise compared to placebo. Palmitate oxidation was increased during submaximal exercise in one patient but did not increase in the two other patients during triheptanoin treatment. Palmitate production and palmitate utilization increased during exercise and increased to a greater extent with triheptanoin treatment in all three patients. This study suggests that triheptanoin treatment has no effect on heart rate or exercise performance despite increased palmitate production and utilization in patients with PFKD.
    Keywords:  Glycogen storage disease; Metabolic Myopathies; Phosphofructokinase deficiency; Triheptanoin; fat and carbohydrate metabolism
    DOI:  https://doi.org/10.1016/j.nmd.2022.01.012
  7. World Rev Nutr Diet. 2022 ;124 351-361
    3.19 Inborn Errors of Metabolism.
    Esther M Maier, Katharina Dokoupil.
      
    Keywords:  Amino acids; Galactosemia; Glycogen storage disease; Maple syrup urine disease; Organic acidurias; Phenylketonuria; Urea cycle disorders
    DOI:  https://doi.org/10.1159/000516735
  8. Orphanet J Rare Dis. 2022 Mar 04. 17(1): 102
    Mild disease course of SARS-CoV-2 infections and mild side effects of vaccination in Pompe disease: a cohort description.
    G Ismailova, M J Mackenbach, J M P van den Hout, A T van der Ploeg, E Brusse, M A E M Wagenmakers.
       INTRODUCTION: Patients with Glycogen Storage Disease type II (GSDII), an inheritable metabolic myopathy also known as Pompe disease, are considered to be at risk for severe COVID-19 due to a reduced respiratory function and a tendency to be overweight. However, so far little is known about the course of SARS-CoV-2 infection and side effects of COVID-19 vaccinations in patients with GSDII.
    METHODS: 169 Dutch Pompe patients are followed at the Erasmus MC Rotterdam. During the COVID-19 pandemic patients were requested to directly inform their physicians about SARS-CoV-2 infection. Infected patients were interviewed regularly by telephone until their symptoms subsided. Furthermore, all patients eligible for vaccination on 16-7-2021 (≥ 17 years, n = 122) were asked to complete a questionnaire.
    RESULTS: To date, fifteen patients (8.9% of our cohort) reported a SARS-CoV-2 infection (classic infantile Pompe disease n = 5, late onset n = 10). No patients were admitted to hospital or needed intensivation of ventilatory support. All patients made a recovery within 19 days. 41.8% of patients filled in our questionnaire regarding vaccination, of whom 98% were vaccinated. Besides one case of perimyocarditis, only mild side effects were reported.
    CONCLUSION: Overall, patients with Pompe disease showed mild symptoms from infection with SARS-CoV-2. All patients made a full recovery. Side effects after vaccination were mostly mild.
    Keywords:  COVID-19; COVID-19 vaccines; Glycogen storage disease type II; Pompe disease; SARS-CoV-2
    DOI:  https://doi.org/10.1186/s13023-022-02268-y
  9. Arthritis Res Ther. 2022 Feb 26. 24(1): 58
    Clinical features of gout in adult patients with type Ia glycogen storage disease: a single-centre retrospective study and a review of literature.
    Na Xu, Xinxin Han, Yun Zhang, Xiaoming Huang, Weiguo Zhu, Min Shen, Wen Zhang, Chen Jialin, Min Wei, Zhengqing Qiu, Xuejun Zeng.
       BACKGROUND: This study aimed to explore the clinical features of gout in adult patients with glycogen storage disease type Ia (GSD Ia).
    METHODS: Ninety-five adult patients with GSD Ia admitted to Peking Union Medical College Hospital were retrospectively analysed. A clinical diagnosis of GSD Ia was confirmed in all patients through gene sequencing. All patients had hyperuricaemia; 31 patients complicated with gout were enrolled, and 64 adult GSD Ia patients with asymptomatic hyperuricaemia were selected as a control group during the same period. Clinical characteristics were analysed and compared between the two groups.
    RESULTS: Thirty-one of the 95 patients had complications of gout (median age, 25 years; 11 (35.5%) females). All 31 patients had hepatomegaly, abnormal liver function, fasting hypoglycaemia, hyperuricaemia, hyperlipaemia, and hyperlacticaemia. A protuberant abdomen, growth retardation, recurrent epistaxis, and diarrhoea were the most common clinical manifestations. Among these 31 patients, 10 patients (32.3%) had gout as the presenting manifestation and were diagnosed with GSD Ia at a median time of 5 years (range, 1-14) after the first gout flare. The median age of gout onset was 18 years (range, 10-29). Fifteen of the 31 GSD Ia-related gout patients were complicated with gouty tophi, which has an average incidence time of 2 years after the first gouty flare. The mean value of the maximum serum uric acid (SUA) was 800.5 μmol/L (range, 468-1068). The incidence of gout in adult GSD Ia patients was significantly associated with the initial age of regular treatment with raw corn starch, the proportion of urate-lowering therapy initiated during the asymptomatic hyperuricaemic stage, maximum SUA level, and mean cholesterol level.
    CONCLUSIONS: Determination of GSD Ia should be performed for young-onset gout patients with an early occurrence of gouty tophi, especially in patients with hepatomegaly, recurrent hypoglycaemia, or growth retardation. Early detection and long-term regulatory management of hyperuricaemia, in addition to early raw corn starch and lifestyle intervention, should be emphasized for GSD Ia patients in order to maintain good metabolic control.
    TRIAL REGISTRATION: Retrospectively registered.
    Keywords:  Glycogen storage disease; Gout; Hyperuricaemia; Type Ia
    DOI:  https://doi.org/10.1186/s13075-021-02706-5
  10. Sheng Wu Gong Cheng Xue Bao. 2022 Feb 25. 38(2): 592-604
    [Engineering the glycogen metabolism in cyanobacterial photosynthetic cell factories: a review].
    Sini Zheng, Huili Sun, Shaoming Mao, Guodong Luan, Xuefeng Lü.
      Cyanobacteria are important photosynthetic autotrophic microorganisms and are considered as one of the most promising microbial chassises for photosynthetic cell factories. Glycogen is the most important natural carbon sink of cyanobacteria, playing important roles in regulating its intracellular carbon distributions. In order to optimize the performances of cyanobacterial photosynthetic cell factories and drive more photosynthetic carbon flow toward the synthesis of desired metabolites, many strategies and approaches have been developed to manipulate the glycogen metabolism in cyanobacteria. However, the disturbances on glycogen metabolism usually cause complex effects on the physiology and metabolism of cyanobacterial cells. Moreover, the effects on synthesis efficiencies of different photosynthetic cell factories usually differ. In this manuscript, we summarized the recent progress on engineering cyanobacterial glycogen metabolism, analyzed and compared the physiological and metabolism effects caused by engineering glycogen metabolism in different cyanobacteria species, and prospected the future trends of this strategy on optimizing cyanobacterial photosynthetic cell factories.
    Keywords:  cyanobacteria; glycogen; metabolic engineering; photosynthesis; photosynthetic cell factory
    DOI:  https://doi.org/10.13345/j.cjb.210230
  11. Reprod Fertil Dev. 2021 Dec;34(2): 272-273
    73 Preimplantation genetic diagnosis of glycogen branching enzyme deficiency and sex determination in equine in vitro-produced embryos.
    M Barandalla, S Colleoni, R Duchi, M Benedetti, A Perota, C Galli, G Lazzari.
      
    DOI:  https://doi.org/10.1071/RDv34n2Ab73
  12. Exp Ther Med. 2022 Mar;23(3): 239
    Combined treatment with niclosamide ethanolamine and artemether combination improves type 1 diabetes via the targeting of liver mitochondria.
    Wenci Weng, Honghong Liu, Zhijian Sun, Peng Zhou, Xuewen Yu, Mumin Shao, Pengxun Han, Huili Sun.
      Type 1 diabetes (T1D) is characterized by dysregulated blood glucose and liver metabolism. In previous studies, niclosamide ethanolamine salt (NEN) and artemether (Art) displayed significant hypoglycemic effects. However, their combined therapeutic effects on the liver in T1D have remained elusive. In the present study, T1D mice were established and randomly allocated into groups. Following treatment, the physiological and metabolic parameters, including liver function, glycogen content, glucose-6-phosphatase (G6Pase) protein expression levels, mitochondrial biogenesis and mitochondrial metabolism were analyzed. Compared with the NEN or Art treatments alone, their combination improved glycometabolism and the symptoms of diabetes. Combined treatment with NEN and Art also significantly ameliorated liver injury and increased liver glycogen storage. Furthermore, combinatorial treatment significantly downregulated hepatic G6Pase protein expression levels and regulated mitochondrial biogenesis. NEN and Art increased the respiratory exchange rate and reduced mitochondrial phosphoenolpyruvate carboxykinase and branched-chain α-keto acid dehydrogenase complex protein expression levels, whereby the effects were obviously enhanced by their application as a combined treatment. In conclusion, the present study confirmed that combined treatment with NEN and Art improved glycometabolism and liver function in T1D mice and the therapeutic effects may be partially associated with the regulation of liver mitochondria.
    Keywords:  artemether; liver; mitochondria; niclosamide ethanolamine; type 1 diabetes
    DOI:  https://doi.org/10.3892/etm.2022.11164
  13. Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2122287119
    Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.
    Traci E LaMoia, Gina M Butrico, Hasini A Kalpage, Leigh Goedeke, Brandon T Hubbard, Daniel F Vatner, Rafael C Gaspar, Xian-Man Zhang, Gary W Cline, Keita Nakahara, Seungwan Woo, Atsuhiro Shimada, Maik Hüttemann, Gerald I Shulman.
      SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.
    Keywords:  biguanides; complex I; complex IV; gluconeogenesis; redox
    DOI:  https://doi.org/10.1073/pnas.2122287119
  14. Ann Nutr Metab. 2021 ;77 Suppl 4 25-27
    Urinary UDP-Glucose as a Novel Actionable Biomarker of Dehydration-Induced Acute Kidney Injury.
    Maria Agustina Battistone, Alexandra C Mendelsohn, Dennis Brown, Sylvie Breton.
       BACKGROUND: People working in "extreme" conditions termed as sugar cane workers, firefighters and military personnel are subjected to significant dehydration. Prolonged episodes of dehydration may result in acute kidney Injury (AKI). AKI is associated with inflammation and is usually diagnosed only after the kidneys have gone through significant and often irreversible damage. We showed that the P2Y14 receptor mediates renal inflammation, leading to AKI following ischemia-reperfusion-injury [<xref ref-type="bibr" rid="ref1">1</xref>]. P2Y14 is activated by the danger molecule UDP-glucose (UDP-Glc). Here we hypothesized that UDP-Glc is released by cells throughout the body after dehydration-induced stress. UDP-Glc is filtered by the kidney and concentrated in collecting ducts where it activates P2Y14 in intercalated cells. This would trigger renal inflammation and contribute to dehydration-associated AKI.
    OBJECTIVE: The aim of this study was to characterize the participation of UDP-Glc in pro-inflammatory cell recruitment and renal dysfunction following dehydration.
    METHOD: Mice were subjected to water deprivation for 24, 48, and 72 h. Kidney function was assessed via serum creatinine (SCr), blood urea nitrogen (BUN), and urine albumin. To study proximal tubule (PT) damage, aquaporin 1 (AQP1) localization was analyzed by immunofluorescence (IF). Urinary UDP-Glc concentration was measured by LC-MS, and renal recruitment of immune cells by flow cytometry and IF.
    RESULTS: Water deprivation induced elevations in SCr and BUN after 48 h and 72 h, relative to control. Dehydration also induced albuminuria and the redistribution of AQP1 from the plasma membrane into the PT cell body indicating PT injury. An increase in urinary UDP-Glc concentration and renal recruitment of macrophages were detected at 48 h and 72 h of dehydration.
    CONCLUSION: This study supports the hypothesis that UDP-Glc, released by damaged cells during severe dehydration, induces the renal recruitment of inflammatory macrophages leading to PT injury and kidney dysfunction (Fig. 1). Blocking the UDP-Glc/P2Y14 pathway represents, therefore, a new therapeutic avenue for the attenuation of dehydration-induced renal inflammation and injury. In this context, urinary UDP-Glc is a promising actionable biomarker for dehydration-induced AKI.
    Keywords:  Acute kidney injury; Dehydration; Inflammation; UDP-glucose
    DOI:  https://doi.org/10.1159/000520566
  15. Anal Chem. 2022 Mar 04.
    In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.
    Noortje de Haan, Yoshiki Narimatsu, Mikkel Koed Møller Aasted, Ida S B Larsen, Irina N Marinova, Sally Dabelsteen, Sergey Y Vakhrushev, Hans H Wandall.
      O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.
    DOI:  https://doi.org/10.1021/acs.analchem.1c05068
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