Name: Spatial Molecular Imaging of the Glycome Using Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2025-11-28T12:40:00.000+00:00
Duration: 8 min 52 s
Description: Learn mass spectrometry imaging for spatial glycobiology using liver tissue samples. This protocol enables reproducible measurement of glycogen and N-linked glycans with enhanced sensitivity.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We acknowledge members of the Vander Kooi, Sun, and Gentry laboratories for fruitful discussions. This study was supported by National Institutes of Health (NIH) grants to the Biospecimen Procurement &#38; Translational Pathology Shared Resource Facility of the University of Kentucky Markey Cancer Center, P30CA177558 to D.B.A., R01AG066653, R01CA266004, R01AG078702, R01CA288696, RM1NS133593 to R.C.S., R35NS116824 to M.S.G., R01DC019054 to C.W.V.K., and the University of Florida College of Medicine.

<ol><li>Caprioli, R. M., Farmer, T. B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+of+biological+samples%3A+Localization+of+peptides+and+proteins+using+MALDI-TOF+MS%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Molecular imaging of biological samples: Localization of peptides and proteins using MALDI-TOF MS.</a> Anal Chem. 69 (23), 4751-4760 (1997).</li>
<li>Buchberger, A. R., Delaney, K., Johnson, J., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+imaging%3A+A+review+of+emerging+advancements+and+future+insights%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Mass spectrometry imaging: A review of emerging advancements and future insights.</a> Anal Chem. 90 (1), 240-265 (2018).</li>
<li>Ouedraogo, R., Daumas, A., Capo, C., Mege, J. L., Textoris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-cell+MALDI-TOF+mass+spectrometry+is+an+accurate+and+rapid+method+to+analyze+different+modes+of+macrophage+activation%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Whole-cell MALDI-TOF mass spectrometry is an accurate and rapid method to analyze different modes of macrophage activation.</a> J Vis Exp. 82, e50926(2013).</li>
<li>Khalil, S. M., Cappuccio, G., Li, F., Maletic-Savatic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+of+human+brain+organoids+using+mass+spectrometry%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Molecular imaging of human brain organoids using mass spectrometry.</a> J Vis Exp. 211, e66997(2024).</li>
<li>Sun, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+glycogen+serves+as+a+critical+glucosamine+cache+required+for+protein+glycosylation%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Brain glycogen serves as a critical glucosamine cache required for protein glycosylation.</a> Cell Metab. 33 (7), 1404-1417 (2021).</li>
<li>Aichler, M., Walch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maldi+imaging+mass+spectrometry%3A+Current+frontiers+and+perspectives+in+pathology+research+and+practice%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">Maldi imaging mass spectrometry: Current frontiers and perspectives in pathology research and practice.</a> Lab Invest. 95 (4), 422-431 (2015).</li>
<li>Conroy, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-dimensionality+reduction+clustering+of+complex+carbohydrates+to+study+lung+cancer+metabolic+heterogeneity%5BTitle%5D)+AND+%22Adv+Cancer+Res%22%5BJournal%5D)">High-dimensionality reduction clustering of complex carbohydrates to study lung cancer metabolic heterogeneity.</a> Adv Cancer Res. 154, 227-251 (2022).</li>
<li>Seeley, E. H., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+of+proteins+in+tissues+by+mass+spectrometry%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Molecular imaging of proteins in tissues by mass spectrometry.</a> Proc Natl Acad Sci U S A. 105 (47), 18126-18131 (2008).</li>
<li>Powers, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix-assisted+laser+desorption+ionization+imaging+mass+spectrometry+workflow+for+spatial+profiling+analysis+of+N-linked+glycan+expression+in+tissues%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Matrix-assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.</a> Anal Chem. 85 (20), 9799-9806 (2013).</li>
<li>Drake, R. R., Powers, T. W., Norris-Caneda, K., Mehta, A. S., Angel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+imaging+of+N-glycans+by+MALDI+imaging+mass+spectrometry+of+fresh+or+formalin-fixed+paraffin-embedded+tissue%5BTitle%5D)+AND+%22Curr+Protoc+Protein+Sci%22%5BJournal%5D)">In situ imaging of N-glycans by MALDI imaging mass spectrometry of fresh or formalin-fixed paraffin-embedded tissue.</a> Curr Protoc Protein Sci. 94 (1), e68(2018).</li>
<li>Stanback, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+n-glycan+and+lipid+analysis+from+tissues+using+MALDI-MS+imaging%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Regional n-glycan and lipid analysis from tissues using MALDI-MS imaging.</a> STAR Protoc. 2 (1), 100304(2021).</li>
<li>Hawkinson, T. R., Sun, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix-assisted+laser+desorption%2Fionization+mass+spectrometry+imaging+of+glycogen+in+situ%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Matrix-assisted laser desorption/ionization mass spectrometry imaging of glycogen in situ.</a> Methods Mol Biol. 2437, 215-228 (2022).</li>
<li>Young, L. E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+mass+spectrometry+imaging+reveals+heterogeneous+glycogen+stores+in+human+normal+and+cancerous+tissues%5BTitle%5D)+AND+%22EMBO+Mol+Med%22%5BJournal%5D)">In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues.</a> EMBO Mol Med. 14 (11), e16029(2022).</li>
<li>Conroy, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+metabolomics+reveals+glycogen+as+an+actionable+target+for+pulmonary+fibrosis%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.</a> Nat Commun. 14 (1), 2759(2023).</li>
<li>Clarke, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycogen+drives+tumor+initiation+and+progression+in+lung+adenocarcinoma%5BTitle%5D)+AND+%22Nat+Metab%22%5BJournal%5D)">Glycogen drives tumor initiation and progression in lung adenocarcinoma.</a> Nat Metab. 7 (5), 952-965 (2025).</li>
<li>Wallace, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+N-glycome+tissue+atlas+of+15+human+normal+and+cancer+tissue+types+determined+by+MALDI+imaging+mass+spectrometry%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">An N-glycome tissue atlas of 15 human normal and cancer tissue types determined by MALDI imaging mass spectrometry.</a> Sci Rep. 14 (1), 489(2024).</li>
<li>Veerasammy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sample+preparation+for+metabolic+profiling+using+MALDI+mass+spectrometry+imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Sample preparation for metabolic profiling using MALDI mass spectrometry imaging.</a> J Vis Exp. 166, e62008(2020).</li>
<li>Mcdowell, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+mass+spectrometry+and+lectin+analysis+of+N-linked+glycans+in+carbohydrate+antigen-defined+pancreatic+cancer+tissues%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Imaging mass spectrometry and lectin analysis of N-linked glycans in carbohydrate antigen-defined pancreatic cancer tissues.</a> Mol Cell Proteomics. 20, 100012(2021).</li>
<li>Palmer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FDR-controlled+metabolite+annotation+for+high-resolution+imaging+mass+spectrometry%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry.</a> Nat Methods. 14 (1), 57-60 (2017).</li>
<li>Colpaert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurological+glycogen+storage+diseases+and+emerging+therapeutics%5BTitle%5D)+AND+%22Neurotherapeutics%22%5BJournal%5D)">Neurological glycogen storage diseases and emerging therapeutics.</a> Neurotherapeutics. 21 (5), e00446(2024).</li>
<li>Taylor, M. J., Lukowski, J. K., Anderton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatially+resolved+mass+spectrometry+at+the+single+cell%3A+Recent+innovations+in+proteomics+and+metabolomics%5BTitle%5D)+AND+%22J+Am+Soc+Mass+Spectrom%22%5BJournal%5D)">Spatially resolved mass spectrometry at the single cell: Recent innovations in proteomics and metabolomics.</a> J Am Soc Mass Spectrom. 32 (4), 872-894 (2021).</li>
<li>Chan, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gel-assisted+mass+spectrometry+imaging+enables+sub-micrometer+spatial+lipidomics%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics.</a> Nat Commun. 15 (1), 5036(2024).</li>
<li>Lim, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maldi+Hiplex-IC%3A+Multiomic+and+multimodal+imaging+of+targeted+intact+proteins+in+tissues%5BTitle%5D)+AND+%22Front+Chem%22%5BJournal%5D)">Maldi Hiplex-IC: Multiomic and multimodal imaging of targeted intact proteins in tissues.</a> Front Chem. 11, 1182404(2023).</li>
<li>Hankin, J. A., Barkley, R. M., Murphy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sublimation+as+a+method+of+matrix+application+for+mass+spectrometric+imaging%5BTitle%5D)+AND+%22J+Am+Soc+Mass+Spectrom%22%5BJournal%5D)">Sublimation as a method of matrix application for mass spectrometric imaging.</a> J Am Soc Mass Spectrom. 18 (9), 1646-1652 (2007).</li>
<li>Rappez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spacem+reveals+metabolic+states+of+single+cells%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Spacem reveals metabolic states of single cells.</a> Nat Methods. 18 (7), 799-805 (2021).</li>
<li>Grgic, A., Cuypers, E., Dubois, L. J., Ellis, S. R., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maldi+MSI+protocol+for+spatial+bottom-up+proteomics+at+single-cell+resolution%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Maldi MSI protocol for spatial bottom-up proteomics at single-cell resolution.</a> J Proteome Res. 23 (12), 5372-5379 (2024).</li>
<li>Dressman, J. W., Bayram, M. F., Angel, P. M., Drake, R. R., Mehta, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+multiomic+MALDI-MSI+analysis+of+lipids+and+N-glycans+through+affinity+array+capture%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Single-cell multiomic MALDI-MSI analysis of lipids and N-glycans through affinity array capture.</a> Anal Chem. 97 (24), 12493-12502 (2025).</li>
<li>Clarke, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+mapping+of+the+brain+metabolome%2C+lipidome%2C+and+glycome%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Spatial mapping of the brain metabolome, lipidome, and glycome.</a> Nat Commun. 16 (1), 4373(2025).</li>
<li>Juras, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+microwave+fixation+provides+an+instantaneous+snapshot+of+the+brain+metabolome%5BTitle%5D)+AND+%22Cell+Rep+Methods%22%5BJournal%5D)">In situ microwave fixation provides an instantaneous snapshot of the brain metabolome.</a> Cell Rep Methods. 3 (4), 100455(2023).</li>
<li>Cantrell, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+high-power%2C+brain-focused+microwave+fixation+to+define+rodent+metabolism%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for high-power, brain-focused microwave fixation to define rodent metabolism.</a> STAR Protoc. 6 (2), 103794(2025).</li>
<li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+AI-driven+framework+to+map+the+brain+metabolome+in+three+dimensions%5BTitle%5D)+AND+%22Nat+Metab%22%5BJournal%5D)">An AI-driven framework to map the brain metabolome in three dimensions.</a> Nat Metab. 7 (4), 842-853 (2025).</li>
</ol>

R.C.S., M.S.G., and C.W.V.K. are co-directors of the Center for Advanced Spatial Biomolecule Research (CASBR) at the University of Florida and are co-founders of Sugar3 LLC. R.C.S. has received research support and/or consultancy fees from Maze Therapeutics. M.S.G. has received research support, research compounds, or consultancy fees from Maze Therapeutics, Valerion Therapeutics, Ionis Pharmaceuticals, PTC Therapeutics, and Aro Biotherapeutics. M.S.G. is a member of the science advisory board for Chelsea's Hope, Glut1-Deficiency Syndrome Foundation, and the Adult Polyglucosan Body Disease Foundation.

MALDI-MSI is an emerging tool for glycome spatial biology. Importantly, while this protocol focuses on data collection using specific hardware, it can be readily adapted to MALDI-MSI platforms from multiple vendors13. Sample processing is identical up until method 4, although some vendors may require the use of specialized slides. Modifications of data collection, using parameters specific to the MALDI-MSI platform, enable rapid adaptation of this protocol. Vendor-specific software for data processing can be utilized, as detailed in step 5, or, alternatively, platforms for rigorous and reproducible MALDI-MSI data processing can be employed19.
MALDI-MSI is a particularly useful tool for understanding biology since different organs, tissues, and cells have different patterns for their glycome. Liver is well-suited for glycome analysis in that it has high levels of glycogen and diverse N-linked glycans13. Other organs have higher levels and diversity of N-linked glycans and lower levels of glycogen13. Importantly, the glycome varies in different organs depending on the metabolic and disease state of the organism, and so the glycome provides a rich source of biological information5,14,15,20. Continuing development of unique carbohydrate-active enzymes for MALDI-MSI analysis promises to extend glycome analysis to additional orthogonal biomolecules in the glycome.
While having many advantages, MALDI-MSI is currently limited in resolution compared to other tools utilized in spatial biology. This protocol utilized 50 &#181;m resolution data collection. While excellent for physiological analysis, data at single-cell resolution would significantly advance the field21. In particular, delocalization can be a significant barrier to increased resolution in methodologies that utilize enzyme spraying and subsequent incubation in humid conditions. Recent advances in sample processing hardware, sample handling methodologies, data acquisition hardware, the use of selective labeling, and data processing are allowing MALDI-MSI data collection to approach cellular resolution22,23. However, there are tradeoffs between resolution and signal-to-noise and inherent limitations based on current sample preparation methods for MALDI that need to be considered. Very specific applications are already achieving the goal of single-cell-resolved MALDI-MSI using innovations in both sample processing24 and novel algorithms, suggesting general solutions to these problems are within reach25,26,27.
Following data collection, the slide can be stained via H&#38;E for the purposes of histological annotation. However, cellular and tissue damage are associated with MALDI-MSI data collection, so this strategy limits the extent of cellular annotation. Alternatively, sequential tissue slices can be utilized for H&#38;E. This strategy provides high-quality data, but requires alignment and integration of discrete sample cuts.
Of note, formalin-fixed paraffin-embedded (FFPE) tissues are very well suited for glycome analysis by MALDI-MSI with small changes in the protocol for sample preparation13. However, FFPE tissue cannot be effectively utilized for metabolomics and other similar analyses, so fresh-frozen samples, as detailed here, are preferred for multiomics approaches28. Sample preparation is a critical component of any MSI pipeline for labile biomolecules, which requires special approaches for harvesting and handling of samples17,29,30.
Methodologies to expand the generality and widespread utilization of MALDI-MSI-based approaches are rapidly progressing with dramatic improvements in hardware and software19. Additionally, recent cutting-edge multiomic28 and multimodal21 approaches combined with machine learning and artificial intelligence are particularly promising for handling these highly complex datasets31.

The field of spatial biology is rapidly expanding, providing critical insights into the dynamic spatial biomolecular complexity of living organisms. Multiple approaches are being employed to study distinct biomolecules in a biological context. While some are highly specialized techniques, mass spectrometry (MS) has emerged as an important general technique to quantify a wide range of biomolecules in the context of cells, tissues, or organisms.
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) is one of the emerging general techniques for multiomic spatial biomolecule imaging. It was initially established for the visualization of proteins and peptides in tissue slices1. Since then, it has been extended to many diverse biological samples and molecules2. The technique can be applied to cellular samples3, organoids4, model animals5, and patient samples6,7. Some molecular classes can be directly measured using MSI-based approaches, including many metabolites and lipids, while other types of more complex biomolecules, such as proteins and glycans, require enzymatic or chemical treatment for them to be suitable for MSI-based detection2,8.
MALDI-MSI has also been applied to the spatial analysis of N-linked glycans and glycogen, utilizing workflows that require enzymatic processing to produce glycan fragments suitable for MSI detection9,10,11,12,13,14,15. N-glycans are cleaved from the asparagine residue of proteins with PNGase F, and glucose chains of glycogen are cleaved with isoamylase. Key advantages of the MALDI glycogen analysis are that it provides spatial definition of glycogen distribution in the biological sample, relative quantification, and information about the branching of the glucose chains. Because both N-glycan and glycogen workflows require enzymatic processing of the sample for MSI analysis, they have been combined into an integrated workflow for glycome analysis (Figure 1A). This workflow requires careful preparation of biological samples and specific processing to enable MS analysis of the glycome, followed by data acquisition and processing. While the workflow requires a series of specialized steps, it produces rich, high-quality spatial glycome data that is highly reproducible and can be adapted to many different types of samples16. Additionally, the workflow is modular, such that the sample processing steps can be performed by independent research laboratories before submission of the samples to specialized facilities that perform the latter steps of MALDI-MSI data acquisition and analysis.

<table><tbody><tr><td>Absorbent underpad</td><td>Denville Scientific</td><td>B1623</td><td></td></tr><tr><td>Acetonitrile (ACN)</td><td>Sigma-Aldrich</td><td>34851-4X4L</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific&amp;nbsp;</td><td>15078292</td><td></td></tr><tr><td>Citraconic anhydride</td><td>Sigma-Aldrich</td><td>125318</td><td></td></tr><tr><td>Conical tube (15mL)</td><td>Alkali Scientific</td><td>CW5600</td><td></td></tr><tr><td>Coplin Jars</td><td>DWK Life Sciences</td><td>900570</td><td></td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica</td><td>CM1860</td><td></td></tr><tr><td>Cryostat chuck</td><td>Leica</td><td>14041926491</td><td></td></tr><tr><td>Desiccator</td><td>Bel Art</td><td>F424004131</td><td></td></tr><tr><td>Dialysis cup</td><td>Thermo Fisher</td><td>88400</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific&amp;nbsp;</td><td>22032601</td><td></td></tr><tr><td>Food steamer</td><td>Rival&amp;nbsp;</td><td>CKRVSTLM21&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Glass insert (70 mm)</td><td>Gel Company</td><td>LGI50</td><td></td></tr><tr><td>Glycogen</td><td>Sigma-Aldrich</td><td>G8876</td><td>Standard</td></tr><tr><td>Horseradish Peroxidase (HRP)</td><td>Sigma-Aldrich</td><td>P8125</td><td>Standard</td></tr><tr><td>HPLC water</td><td>Sigma-Aldrich</td><td>270733-4X4L</td><td></td></tr><tr><td>HTX Sprayer</td><td>HTX Technologies</td><td>M5</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific&amp;nbsp;</td><td>A144-500</td><td></td></tr><tr><td>Isoamylase</td><td>Megazyme</td><td>E-ISAMY</td><td></td></tr><tr><td>Knauer pump</td><td>Knauer</td><td>P 4.1 S</td><td></td></tr><tr><td>Low-profile blade</td><td>Electron Microscopy Sciences</td><td>63059-01</td><td></td></tr><tr><td>M1 mounting media</td><td>Epredia</td><td>1310</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Bruker Daltonics</td><td>timsTOF fleX</td><td></td></tr><tr><td>Microfuge tube (1.5mL)</td><td>Alkali Scientific</td><td>CN3016</td><td></td></tr><tr><td>Needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Neutral buffered formalin NBF</td><td>Millipore sigma</td><td>HT501128-4L</td><td></td></tr><tr><td>Nitrogen Gas</td><td>Airgas</td><td>NIUHP300</td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific&amp;nbsp;</td><td>SH30256.FS</td><td></td></tr><tr><td>PCR tubes</td><td>MedSupply Partners</td><td>62-1091-1</td><td></td></tr><tr><td>PNGase F PRIME-LY ULTRA&amp;nbsp;</td><td>Bulldog Bio</td><td>NZPULT10LY</td><td></td></tr><tr><td>SCiLS Lab</td><td>Bruker Daltonics</td><td>1889000</td><td></td></tr><tr><td>Shaker</td><td>Thermo Scientific</td><td>88882007</td><td></td></tr><tr><td>Slide&amp;nbsp;&amp;nbsp;</td><td>Electron Microscope Sciences</td><td>71873-02</td><td></td></tr><tr><td>Slide mailer</td><td>RPI products</td><td>212620A</td><td></td></tr><tr><td>Sonicater (Bioruptor Pico)</td><td>Diagenode</td><td>B01080010</td><td></td></tr><tr><td>Sterile Forceps</td><td>DR Instruments</td><td>S08098</td><td></td></tr><tr><td>Sterile Scalpel</td><td>WPI</td><td>WPI-500240</td><td></td></tr><tr><td>Steriles Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Syringe&amp;nbsp;&amp;nbsp;</td><td>Grainger</td><td>19G342</td><td></td></tr><tr><td>Syringe pump</td><td>New Era</td><td>NE-300</td><td></td></tr><tr><td>Tape</td><td>Fisher Scientific&amp;nbsp;</td><td>15-901-R</td><td></td></tr><tr><td>Thin paintbrush</td><td>Anezus</td><td>#00</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508-1L</td><td></td></tr><tr><td>Tune Mix, ESI low concentration</td><td>Agilent</td><td>G1969-85000</td><td></td></tr><tr><td>Weight boats</td><td>Sigma-Aldrich</td><td>HS120882</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>534056</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid (CHCA)</td><td>Cayman Chemical Company</td><td>15254</td><td></td></tr></tbody></table>

spatial molecular imaging of the glycome using mass spectrometry

Spatial Molecular Imaging of the Glycome Using Mass Spectrometry

The spatial organization of the glycome within tissues is key to the molecular basis for physiological function. The diverse and dynamic glycome is critical for fundamental cellular processes, including metabolism, signaling, and adhesion. Innovations in spatial biology have ushered in new avenues for spatial molecular imaging of diverse glycome classes. Here, we describe an optimized protocol for spatial biomolecular imaging of the glycome in fresh-frozen mouse liver. The workflow comprises (1) rapid harvesting and freezing, (2) cryostat sectioning at optimal thickness and position, (3) tissue preparation and on-tissue enzyme digestion using carbohydrate-active enzymes, (4) matrix application and data acquisition by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and (5) data processing and visualization to place the findings in biological context. Use of this approach allows acquisition of detailed spatial maps of N-linked glycans and glycogen, revealing key physiological and cellular features. These data allow the definition of key spatial glycomic heterogeneity associated with liver function and dysfunction. This workflow enables highly reproducible and sensitive spatial glycomics of the mouse liver. Additionally, it is readily adaptable to other tissues or species, facilitating novel spatial insights into glycome biology in health and disease.

Careful processing of a liver from a wild-type mouse, including cutting a full transverse section (Figure 1A), allowed definition of the glycome by MALDI-MSI, with diverse glycogen-derived oligosaccharides and N-linked glycans over a range of m/z (Figure 1B). Glycogen was broadly localized throughout the liver hepatocytes and lacking in vessels and connective tissue in the portal tracts, as expected (Figure 1C,D). Importantly, the MALDI-MSI data allowed measurement of both the spatial distribution of glycogen as well as chain-length distribution, a key feature of glycogen in normal metabolism and disease states (Figure 1D). Additionally, diverse N-linked glycans exhibit diverse organizations. The observed spatial distribution ranges from widely distributed in hepatocytes (Figure 1E) to regional foci, consistent with the sinusoidal organization of the liver (Figure 1F), to specific labeling of portal tract elements (Figure 1G-I).
<img alt="Mass spectrometry workflow diagram, spectral data and liver tissue imaging, showing glycan analysis results." class="xfigimg" src="/files/ftp_upload/69154/69154fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: MALDI-MSI glycome analysis of mouse liver. (A) Schematic for critical methods in sample preparation, data acquisition, and analysis. (B) Sample mass spectra derived from the approach. Arrows in red demonstrate the regularly spaced oligosaccharides derived from glycogen, differing by one glucose monosaccharide. Arrows in blue denote N-glycans, which are found distributed throughout the spectra. In the liver, glycogen peaks are generally more prominent, especially at higher m/z values. (C) Spatial distribution of maltohexaose (DP6) derived from glycogen in the liver. (D) Distribution of glycogen chain lengths in the liver. (E-G) Spatial distribution of different N-linked glycan species in the liver. (H) Hematoxylin and Eosin (H&amp;E) staining for a focused central region, along with anatomical annotations. (I) Corresponding focused inset showing the spatial localization of additional N-linked glycan (Hex5dHex1HexNAc5) in portal tract elements. Scale bars: 500 µm. <a href="https://www.jove.com/files/ftp_upload/69154/69154fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol details the key steps to enable rigorous and reproducible measurement of both glycogen and N-linked glycans using mass spectrometry imaging.

The spatial organization of the glycome within tissues is key to the molecular basis for physiological function. The diverse and dynamic glycome is ...

We are using exciting, new mass spectrometry-based technologies to map biomolecules in biological samples for spatial biology. Key current challenges include rigor and reproducibility coming from sample preparation and limitations in sensitivity and resolution. To begin, prepare one or more dialysis cups by placing them in high performance liquid chromatography or HPLC water for 15 minutes.Dialyze 200 microliters of stock isoamylase using a 500 microliter dialysis cup in 14.3 milliliters of HPLC water for two hours at four degrees Celsius without stirring. Using a pipette, gently remove the isoamylase from the dialysis cup and place it into a 1.5 milliliter micro centrifuge tube. Measure the total volume of the isoamylase solution after dialysis.Based on the stock activity of 200 units per milliliter and the final volume, calculate the total volume required for three units of isoamylase. Pipette aliquots containing three units of isoamylase into thin-walled PCR tubes. Snap freeze the tubes in liquid nitrogen and store them at minus 80 degrees Celsius.When tissues are processed and ready, prepare the humidity chamber by placing a paper towel soaked with deionized water below the metal rack. Place the chamber in a 37 degrees Celsius incubator for equilibration. Then fill the food steamer reservoir with tap water to the top and set the timer to 60 minutes to turn the unit on.Place the slide with the tissue facing inward to avoid contact with the sides and to ensure effective antigen retrieval. Fill each slide mailer with the freshly prepared citraconic buffer. Verify that steam is being emitted from the food steamer.Place the mailers in the steamer and incubate them for 30 minutes. Then transfer the mailers to a deionized water bath for five minutes. Replace half of the citraconic buffer in each mailer with deionized water and allow it to stand for another five minutes.Remove all solvent and wash the slides by adding 100%deionized water to each mailer, and then removing it. On the back of each slide, draw a circle and a triangle to mark internal standard locations. Apply one microliter of one milligram per milliliter horse radish peroxidase at the circle mark, and one microliter of 10 milligrams per milliliter rabbit liver glycogen at the triangle mark.Thaw one tube of 100 micrograms of lyophilized peptide N Glycosidase F and one tube containing three units of isoamylase for 10 minutes. Centrifuge the tubes at approximately 2000 G for five seconds. Pipette 50 microliters of deionized water into each lyophilized PNGase F tube.After vortexing gently, centrifuge the tube at approximately 2000 G for five seconds. Pipette the resulting 50 microliters of PNGase F solution directly into the isoamylase tube and add HPLC water to bring the total volume to one milliliter. Turn on the HTX M5 sprayer and open the HTX M5 software on the connected computer.Turn off the Knauer pump by pressing the stop button on the front right side and power on the external syringe pump using the switch on the back panel. In the HTX software, under the Methods tab, select the appropriate method for dual enzyme spraying. Navigate to the Temp tab and set the spray nozzle temperature to 45 degrees Celsius.Open the ultra-high purity nitrogen gas valve and ensure the sprayer pressure is set to 10 pounds per square inch. Using a fresh syringe and needle, fill it with four milliliters of HPLC-grade water. After removing the bubbles, run two milliliters of water through approximately a six inch section of the sprayer line into a waste beaker.Attach the syringe to the sprayer line and run water through the syringe pump by starting the system at a flow rate of 95 microliters per minute for five minutes. While the syringe pump is flushing, tape the prepared slides to the bottom left corner of the heated tray using a metal alignment guide. Under the Sample tab, define the X and Y parameters for the spray region.After five minutes, pause the syringe pump and disconnect the syringe. Draw air into it and push the air through the sprayer line to purge any residual water. Then load the prepared dual enzyme solution into a new syringe and connect it securely to the syringe pump.Attach the loaded syringe containing the dual enzyme solution to the sprayer line. Once the spray nozzle temperature reaches 45 degrees Celsius, press Start under the Cycle tab. When prompted to turn on the Knauer pump, click No.Press start on the syringe pump to begin enzyme spraying.Place a blank slide under the spray nozzle to confirm visible enzyme deposition. Once the enzyme spray is visibly even, click continue on the software. Next, place the glass cover on the sprayer and monitor for even application, ensuring uniform wetting across the slide surface.When the spray cycle is complete, press stop on the syringe pump. Turn off the nitrogen gas valve and click Valve Load Confirm in the software. Incubate the sprayed slides in the HTX humidity chamber at 37 degrees Celsius for two hours, ensuring the slides are placed facing upward.Once incubation is complete, transfer the slides to a desiccator and dry for 15 minutes. Glycogen-derived oligosaccharides and N-linked glycans were detected across a wide master charge range in the liver using matrix-assisted laser desorption ionization mass spectrometry imaging. Glycogen was broadly localized throughout liver hepatocytes while it was absent from vessels and connective tissue in the portal tracts.The spatial distribution of glycogen and its chain length distribution were both measurable using MALDI MSI. One N-Linked glycan species was widely distributed throughout liver hepatocytes. Another N-linked glycan species displayed a more regional distribution pattern consistent with liver sinusoidal structure.A distinct and linked glycan localized specifically to portal tract elements. Histological staining confirmed the anatomical structures of the portal tract, including portal vein, bile duct, and connective tissue. We utilize spatial molecular imaging to uncover the cellular, metabolic and molecular underpinnings of cellular biology, physiology, and disease pathology.These findings will allow molecular imaging of the glycome in diverse biological sources and disease states. Future research will allow detection of different glycome classes and increase the sensitivity and resolution to a single-cell level.

spatial-molecular-imaging-of-the-glycome-using-mass-spectrometry

Research

JoVE Journal

Biochemistry

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Mass Spectrometric Analysis of Glycosphingolipid Antigens

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Glycan Node Analysis: A Bottom-up Approach to Glycomics

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Here, we present a detailed protocol for the release of O-glycans from mucins, and the subsequent desalting, permethylation, and analysis using MALDI-TOF mass spectrometry.

Profiling of Permethylated Mucin O-glycans Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

A protocol was developed for the preparation of purified mitochondria from microglial cells, isolation of mitochondrial proteins for N-glycan release, and rapid detection of subcellular, mitochondrial glycans using infrared matrix-assisted laser desorption electrospray ionization coupled to high-resolution accurate mass analyzer mass spectrometry.

Mitochondrial Preparation from Microglia for Glycan Analysis

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Learn mass spectrometry imaging for spatial glycobiology using liver tissue samples. This protocol enables reproducible measurement of glycogen and N-linked glycans with enhanced sensitivity.

Spatial Molecular Imaging of the Glycome Using Mass Spectrometry

Explore More Videos