In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues
2022; Springer Nature; Volume: 14; Issue: 11 Linguagem: Inglês
10.15252/emmm.202216029
ISSN1757-4684
AutoresLyndsay E.A. Young, Lindsey R. Conroy, Harrison A. Clarke, Tara R. Hawkinson, Kayli E. Bolton, William C. Sanders, Josephine E. Chang, Madison B. Webb, Warren J. Alilain, Craig W. Vander Kooi, Richard R. Drake, Douglas Andres, Tom C. Badgett, Lars M. Wagner, Derek B. Allison, Ramon C. Sun, Matthew S. Gentry,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoArticle5 September 2022Open Access Transparent process In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues Lyndsay E A Young Lyndsay E A Young orcid.org/0000-0002-6873-5674 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Lindsey R Conroy Lindsey R Conroy Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Harrison A Clarke Harrison A Clarke Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation Search for more papers by this author Tara R Hawkinson Tara R Hawkinson orcid.org/0000-0002-8743-5503 Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Methodology Search for more papers by this author Kayli E Bolton Kayli E Bolton orcid.org/0000-0002-5376-4325 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author William C Sanders William C Sanders Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Josephine E Chang Josephine E Chang Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Madison B Webb Madison B Webb Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Warren J Alilain Warren J Alilain orcid.org/0000-0003-4502-8878 Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Craig W Vander Kooi Craig W Vander Kooi Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Richard R Drake Richard R Drake Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA Contribution: Resources, Investigation, Methodology Search for more papers by this author Douglas A Andres Douglas A Andres Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources, Investigation Search for more papers by this author Tom C Badgett Tom C Badgett Pediatric Hematology-Oncology, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources Search for more papers by this author Lars M Wagner Lars M Wagner orcid.org/0000-0003-4717-9960 Pediatric Hematology-Oncology, Duke University, Durham, NC, USA Contribution: Resources Search for more papers by this author Derek B Allison Derek B Allison orcid.org/0000-0002-5119-2474 Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources, Formal analysis, Investigation Search for more papers by this author Ramon C Sun Corresponding Author Ramon C Sun [email protected] orcid.org/0000-0002-3009-1850 Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, USA Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA Center for Advanced Spatial Biomolecule Research, University of Florida, Gainesville, FL, USA Contribution: Conceptualization, Resources, Supervision, Writing - original draft, Project administration Search for more papers by this author Matthew S Gentry Corresponding Author Matthew S Gentry [email protected] orcid.org/0000-0001-5253-9049 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA Center for Advanced Spatial Biomolecule Research, University of Florida, Gainesville, FL, USA Contribution: Conceptualization, Resources, Writing - original draft, Project administration Search for more papers by this author Lyndsay E A Young Lyndsay E A Young orcid.org/0000-0002-6873-5674 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Lindsey R Conroy Lindsey R Conroy Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Harrison A Clarke Harrison A Clarke Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation Search for more papers by this author Tara R Hawkinson Tara R Hawkinson orcid.org/0000-0002-8743-5503 Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Investigation, Methodology Search for more papers by this author Kayli E Bolton Kayli E Bolton orcid.org/0000-0002-5376-4325 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author William C Sanders William C Sanders Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Josephine E Chang Josephine E Chang Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Madison B Webb Madison B Webb Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Warren J Alilain Warren J Alilain orcid.org/0000-0003-4502-8878 Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, USA Contribution: Investigation Search for more papers by this author Craig W Vander Kooi Craig W Vander Kooi Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Richard R Drake Richard R Drake Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA Contribution: Resources, Investigation, Methodology Search for more papers by this author Douglas A Andres Douglas A Andres Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources, Investigation Search for more papers by this author Tom C Badgett Tom C Badgett Pediatric Hematology-Oncology, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources Search for more papers by this author Lars M Wagner Lars M Wagner orcid.org/0000-0003-4717-9960 Pediatric Hematology-Oncology, Duke University, Durham, NC, USA Contribution: Resources Search for more papers by this author Derek B Allison Derek B Allison orcid.org/0000-0002-5119-2474 Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky, Lexington, KY, USA Contribution: Resources, Formal analysis, Investigation Search for more papers by this author Ramon C Sun Corresponding Author Ramon C Sun [email protected] orcid.org/0000-0002-3009-1850 Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, USA Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA Center for Advanced Spatial Biomolecule Research, University of Florida, Gainesville, FL, USA Contribution: Conceptualization, Resources, Supervision, Writing - original draft, Project administration Search for more papers by this author Matthew S Gentry Corresponding Author Matthew S Gentry [email protected] orcid.org/0000-0001-5253-9049 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA Markey Cancer Center, University of Kentucky, Lexington, KY, USA Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA Center for Advanced Spatial Biomolecule Research, University of Florida, Gainesville, FL, USA Contribution: Conceptualization, Resources, Writing - original draft, Project administration Search for more papers by this author Author Information Lyndsay E A Young1,2,†, Lindsey R Conroy2,3,†, Harrison A Clarke3, Tara R Hawkinson3, Kayli E Bolton1, William C Sanders1, Josephine E Chang3, Madison B Webb1, Warren J Alilain3,4, Craig W Vander Kooi1,2, Richard R Drake5, Douglas A Andres1, Tom C Badgett6, Lars M Wagner7, Derek B Allison8, Ramon C Sun *,2,3,4,9,10 and Matthew S Gentry *,1,2,9,10 1Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA 2Markey Cancer Center, University of Kentucky, Lexington, KY, USA 3Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA 4Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, USA 5Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA 6Pediatric Hematology-Oncology, College of Medicine, University of Kentucky, Lexington, KY, USA 7Pediatric Hematology-Oncology, Duke University, Durham, NC, USA 8Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky, Lexington, KY, USA 9Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA 10Center for Advanced Spatial Biomolecule Research, University of Florida, Gainesville, FL, USA † These authors contributed equally to this work *Corresponding author. Tel: +352 294 8407; E-mail: [email protected] *Corresponding author. Tel: +352 294 8387; E-mail: [email protected] EMBO Mol Med (2022)14:e16029https://doi.org/10.15252/emmm.202216029 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma. Synopsis Development of a MALDI-based assay for the spatial quantification of microenvironmental glycogen and glycogen biochemical architecture. Hyperphosphorylated glycogen was discovered in human Ewing sarcoma. Targeting tumor-specific glycogen may be a potential therapeutic approach for Ewing sarcoma. Development of a MALDI-based assay for the spatial quantification of microenvironmental glycogen. Ultra-sensitivity allows visualization of glycogen in previously unknown but distinct cellular layers in multiple human tissues. Identification of glycogen-rich and glycogen-poor tumors such as Ewing sarcoma and prostate cancer, respectively. Targeting Ewing sarcoma glycogen by different modalities blunted tumor growth in immunodeficient mice. The paper explained Problem Physiological glycogen levels often fall below the detection limit of current histopathological methodologies. Due to the technical gap in the tools available, glycogen heterogeneity and spatial distribution in healthy and diseased tissues remains largely unknown as does the role of excess glycogen in driving pathogenesis. Development of a new method that combines glycogen architectural information with spatial distribution would be a major advancement to aid in our understanding of glycogen metabolism in both normal and disease conditions. Results Herein, we introduce a robust and sensitive workflow that provides deep interrogation of glycogen content, architecture, and spatial distribution in an array of healthy and diseased mammalian tissues, including several human cancers. Armed with this new tool, we demonstrate that glycogen levels are heterogeneous among both healthy and diseased tissues. Importantly, we identify structurally unique glycogen as a clinical feature of the pediatric cancer, Ewing sarcoma. Furthermore, we demonstrate the therapeutic potential of targeting the unique glycogen in Ewing sarcoma preclinical models. Impact Collectively, our workflow provides a sensitive and precise method to interrogate the spatial glycogen architecture and localization in situ. Furthermore, our data support aberrant glycogen as a clinical hallmark of Ewing sarcoma and highlight multiple therapeutic entry points for drug discovery against glycogen for the treatment of this pediatric cancer. Introduction Glycogen is both an intracellular metabolite and a macromolecule with a mass that can be altered by several orders of magnitude via release of glucose-1-phosphate upon extracellular stimuli (Persson et al, 2020). Over a hundred years of glycogen-centric research has established foundational concepts regarding: metabolism (Bernard, 1857), protein structure–function (Fischer & Krebs, 1955), and intracellular signaling (Sun et al, 2019; Liu et al, 2021). Glycogen is metabolically dynamic (Prats et al, 2018) and can be directly channeled to other metabolic processes including glycolysis and the Krebs cycle for ATP production (Nordlie et al, 1999). Administration of 13C-glucose in human volunteers demonstrated glucose flux through glycogen in minutes under nonfasting conditions, suggesting that active glycogen synthesis and glycogenolysis play unknown (yet-to-be discovered) roles in organismal physiology (Oz et al, 2007). A recent study revealed glycogen as the major contributor to glycolytic intermediates in most major organs under physiological conditions (TeSlaa et al, 2021). In addition, glycogen supplies metabolite pools for unique cellular processes. Glycogen metabolism has recently been linked to the modulation of epigenetics via nuclear glycogenolysis to supply acetyl-CoA (Sun et al, 2019), and liquid-phase separation of protein-bound glycogen is a driver of both liver tumorigenesis and proliferation (Liu et al, 2021). Glycogen is comprised of α-1,4- and α-1,6-linked linear glucose polymers that enable maximum packing efficiency (Roach, 2002). Glycogen biosynthesis is achieved through the stepwise actions of glycogen synthase (GYS), forming α-1,4-glycosidic linkages, and branching enzyme (BE), adding α-1,6-glycosidic linkages every 10–15 glucose residues. Additionally, phosphate is covalently attached at glucose hydroxyls during synthesis (Roach, 2015). Glucose is released from glycogen by the actions of glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE; Brewer & Gentry, 2019). Glycogen architecture encompasses the modeling of α-1,6-branches, glucose chain length, and total phosphate esters, which are modulated by the glycogen phosphatase laforin (Worby et al, 2006; Adeva-Andany et al, 2016). Together, these architectural parameters define the granular size, crystallinity, and solubility of a glycogen molecule within a cell. For example, glucose chain length directly impacts formation of large (50–100 μm), pathogenic glycogen aggregates also known as polyglucosan bodies (PGBs). PGB formation and phase separation render PGBs inaccessible to enzymes (Sullivan et al, 2017; Persson et al, 2020; Liu et al, 2021). PGBs have also been identified in the pleural perfusion of the lung (Röcken et al, 1996), aging prostate (Röcken et al, 1996), Parkinson's disease (Riba et al, 2019), Alzheimer's disease (Riba et al, 2019), and many types of cancers (Christian et al, 2005). However, the disease-specific pathological roles of these glycogen-like aggregates and differences in their architecture remain to be defined. Purified glycogen can be assessed using chromatography-based methods to quantify glucose chain length and phosphate levels, but these methods lack sensitivity, tissue-specific spatial resolution, and structural information (Young et al, 2020). Of note, periodic acid-Schiff (PAS) is used to assess spatial glycogen; however, due to low specificity and sensitivity, its application is limited to the liver, muscle, and certain types of high glycogen cancers (Aterman & Norkin, 1963). Glycogen stores and architecture can change dramatically in response to stimuli or microenvironmental changes during exercise and disease. Additionally, glycogen levels vary dramatically among cell types and subtissue regions. Therefore, a new method that combines architectural information with spatial distribution and the sensitivity of mass spectrometry would be a major advancement to aid in our understanding of glycogen metabolism in both physiological and disease conditions. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is at the forefront of technological innovations while being implemented to make significant clinical and biological discoveries in the last decade (Powers et al, 2015). Enzyme-assisted MALDI-MSI is a relatively new technique that can release a quantifiable metabolic product, enabling the utilization of the vast amount of clinical resources stored in the form of formalin-fixed paraffin-embedded (FFPE) tissues (Clift et al, 2021; Conroy et al, 2021). We previously demonstrated the early application and potential of MALDI-MSI to visually quantify glycogen in situ (Hawkinson & Sun, 2022). Herein, we introduce a comprehensive workflow using ion-mobility MALDI-MSI for unambiguous and deep interrogation of glycogen levels, architecture, and spatial origin in mammalian tissues. Armed with this new tool, we report architecturally unique glycogen localization in an array of mouse and human healthy and diseased tissues. Most interestingly, we identified excess PGBs in the pediatric cancer, Ewing sarcoma. Furthermore, we demonstrate the therapeutic potential of PGBs in Ewing sarcoma using two different modalities targeting PGBs that ablated xenograft tumor growth in vivo. Results Enzyme-assisted MALDI imaging of spatial glycogen in situ MALDI imaging has been employed for the spatial profiling of N-linked glycans from FFPE tissues after enzymatic hydrolysis of N-linked glycans from proteins using peptide-N-glycosidase F (PNGase F) with spatial details ranging from macro-to micro-tissue structures (Drake et al, 2018; Stanback et al, 2021). We hypothesized that a similar approach could be adapted for the spatial profiling of glycogen in tissues. For the enzymatic digestion of glycogen, we employed isoamylase (Glycogen 6-glucanohydrolase, Megazyme), a bacterial enzyme that specifically cleaves the glycogen α-1,6-glycosidic bonds to release linear glucose polymers from glycogen that range 3–25 glucose units in length (Harada et al, 1972; Fig 1A). To test the utility of isoamylase in MALDI-MSI, purified rabbit liver glycogen was directly spotted and dried on a microscope slide, processed through an antigen retrieval step, and then isoamylase (3 U) was applied using a high-velocity dry sprayer to cleave glycogen into glucose polymers. Finally, α-cyano-4-hydroxycinnamic acid (CHCA) ionization matrix was applied using the same dry sprayer with modified parameters (Fig 1A). Released glucose polymers were analyzed by MALDI mass spectrometry using a Waters Synapt G2 XS ion mobility-enabled mass spectrometer equipped with an Nd:YAG laser. Two-hour isoamylase digestion generated stepwise peaks that are 162 m/z apart as recorded by the time-of-flight (TOF) mass detector (Fig 1B). 162 m/z corresponds to the 1 glucose unit difference among polymers and agrees with previously published glucose polymer patterns established by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAC-PAD; Hanashiro et al, 1996). Figure 1. In situ digestion of glycogen by isoamylase releases glucose polymers, detectable by MALDI-MSI A. (Top) Schematic of MALDI-MSI workflow: FFPE tissue slices (4 μm) processed through antigen retrieval, enzyme digestion, and matrix application followed by ionization by argon laser and detection by time-of-flight (TOF) detector. (Bottom) Schematic of isoamylase digestion of glycogen from tissue sections cleaving alpha-1,6-glycosidic bonds, releasing linear glucose polymers. B. Representative ion chromatogram of linear glucose polymers (GP) detected by MALDI-TOF following isoamylase digestion of (top) purified rabbit liver glycogen and (bottom) mouse liver tissue. GP4 to GP13 and their masses (rounded to the nearest one) were highlighted for better visualization. C. Spatial distribution of unique GP4-8 from a wild-type (WT) mouse liver. The image displays a heatmap with gradient of intensity from white (least abundant) to red (most abundant). Scale bar is represented below the images. D. Schematic of a multiplex analysis of glucose polymers and N-linked glycans after PNGase F and isoamylase digestion followed by traveling wave ion mobility separation (TW-IMS). E. Scatter plot of monoisotopic mass versus drift time in the ion mobility cell for glucose polymers and N-glycans from mouse liver tissue. F. Hematoxylin and eosin (H&E)-stained cross section of a mouse liver. Scale bar is representative of both (F) and (G). Image is also used in Fig 2A. G. Overlay MALDI-IMS image of glycogen (CL7, m/z = 1,175 blue) and N-linked glycans (m/z = 1,663 green; m/z = 1,809 red) of an immediate adjacent slice to the H&E section of (F). H. Relative abundance of m/z extracted from the glucose polymer regions of (E) representing glycogen chain lengths ranging from m/z = 500–3,500. I. Relative abundance of m/z extracted from the glycan regions of (E) representing N-linked glycans from m/z = 500–3,500. Download figure Download PowerPoint Glycogen phosphorylation is a critical biochemical modification regulated by the glycogen phosphatase laforin (Worby et al, 2006; Tagliabracci et al, 2007). Phosphate can be covalently linked to glycogen at the C2-, C3-, and C6-hydroxyl residues of glucose (Appendix Fig S1A; Tagliabracci et al, 2011). Glycogen hyperphosphorylation results in PGBs that impact glycogen turnover, and perturbations in this process have deleterious consequences as mutations in the gene encoding laforin result in the fatal childhood dementia and progressive myoclonus epilepsy Lafora disease (Turnbull et al, 2016; Gentry et al, 2018). Phosphorylated glycogen is difficult to quantify using conventional biochemical methods. In addition to glucose polymers, we were also able to identify phosphorylated glucose polymers (Appendix Fig S1B). Thus, we can characterize multiple parameters of glycogen architecture, that is, chain length and phosphate levels, using MALDI mass spectrometry. To test whether this method can be applied to whole tissue, we processed FFPE C57BL/6J mouse liver through the workflow. Liver is the most well-known site of glycogen storage and plays a key role in regulating whole-body blood glucose concentration (Hultman & Nilsson, 1971). FFPE 3-month-old mouse liver was sectioned at 4 μm thickness followed by sequential application of isoamylase and CHCA by high-velocity dry spraying and analyzed by MALDI-MSI. Mouse liver exhibited a similar glucose polymer distribution pattern compared with purified glycogen (Fig 1B). The glycogen spatial distribution within the liver was generated using the relative abundance of the most prominent glucose polymers, which are 4–8 glucose units in length (Fig 1C). To test whether the workflow is robust across different MALDI-MSI platforms, an adjacent section of liver tissue was scanned for glucose polymer distribution using a Bruker TIMS-TOF Flex instrument after parallel treatment by isoamylase and application of CHCA at the Medical University of South Carolina. We observed nearly identical chain length distribution pattern and glycogen regional distribution between two different platforms (Appendix Fig S1C and D). These data confirm that the method is robust and reproducible across institutions and different mass spectrometer platforms. Traveling wave ion mobility separation is a relatively new technology that provides de novo separation of molecular ions with similar m/z but different collision cross section (Shvartsburg & Smith, 2008). We hypothesized that N-linked glycans and glycogen could be multiplexed in one assay with the aid of ion mobility separation. To test this hypothesis, we performed co-spraying of PNGase F and isoamylase (Fig 1D) and incubated the slide for 2 h followed by CHCA matrix application and MALDI-MSI analysis. As predicted, co-treatment of PNGase F and isoamylase produced both N-linked glycans and glycogen-derived glucose polymers (Fig 1E–G and Appendix Fig S1E–G) that displayed differential migration through the traveling wave ion mobility chamber (Fig 1E, H and I, and Appendix Fig S1E–G). Multiplexed imaging of both N-linked glycans and glycogen revealed distinct spatial differences through the cross section of WT mouse liver (Fig 1G). Thus, this MALDI-MSI multiplexed workflow provides quantification of both N-linked glycans and glycogen while also providing spatial distribution throughout the tissue. Heterogeneous glycogen spatial distribution in major organs of C57BL/6J mice Glycogen has been reported in multiple tissues in both mice and humans (Zois et al, 2014; Adeva-Andany et al, 2016). However, a detailed simultaneous spatial distribution and architectural assessment remain a critical knowledge gap in understanding the biological roles of glycogen in these tissues. We applied the MALDI-MSI method to examine glycogen composition in multiple wild-type C57BL/6J mouse tissues. First, we performed spatial glycogen analysis of the mouse liver. Heatmap distribution of the most abundant glucose polymer ion, DP 7, exhibits heterogeneous localization of glycogen in the mouse liver. Strikingly, the connective tissue layer encapsulating the liver known as the Glisson's capsule (GC) and the endothelium lining (EL) of the central vein have significantly higher levels of glycogen compared with hepatocytes (H) (Fig 2A–C). Furthermore, there was a gradient of glycogen stores radiating away from the central vein (EL) as evidenced by pixel analyses from three separate regions of the tissue cross section (Appendix Fig S2A). The relative abundance of each glucose polymer recorded by MALDI-MSI also contains data regarding glycogen chain length, a crucial architectural parameter of glycogen. MALDI-MSI analysis of glycogen chain length suggested a major decrease in glycogen occurs between chain length 4 and 8 between the histological regions (Fig 2D), while no significant decrease between chain length 9 and 18 among different anatomical regions of the mouse liver. Figure 2. Spatial ana
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