Optimized Protocol for MALDI MSI of N-glycans Using an On-tissue Digestion in Fresh Frozen Tissue Sections

Overview

Journal Sci Rep

Specialty Science

Date 2023 Feb 16

PMID 36797298

Authors

Andrej Grgic

Kasper K Krestensen

Ron M A Heeren

Affiliations

Soon will be listed here.

Abstract

Glycans play an important role in biology with multiple cellular functions ranging from cell signaling, mobility and growth to protein folding and localization. The N-glycosylation state within a tissue has been found to vary greatly between healthy and diseased patients and has proven to have an important clinical diagnostic value. Matrix assisted laser-desorption ionization (MALDI) mass spectrometry imaging (MSI) allows for untargeted analysis of biomolecules, including N-glycans, on a tissue section and provides a spatial context of the analyte. Until now, N-glycans have been predominantly analyzed using MALDI MSI on formalin-fixed paraffin embedded (FFPE) tissue sections, however this greatly reduces the clinical applicability, as the FFPE embedding process alters the biological environment of the tissue. Here we developed a protocol that allows for MALDI MSI of N-glycans from fresh frozen tissue that matches the current standard of FFPE analysis. By optimizing several steps in the sample preparation, we see orders of magnitude increase in signal intensity. Furthermore, this method limits delocalization of released N-glycans, thus improving the effective spatial resolution of the label-free molecular images. This protocol provides a novel perspective towards clinical application of MALDI MSI and capitalizes on the diagnostic value of N-glycan analysis.

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References

Patterson M . Metabolic mimics: the disorders of N-linked glycosylation. Semin Pediatr Neurol. 2006; 12(3):144-51. DOI: 10.1016/j.spen.2005.10.002. View

Velickovic D, Sharma K, Alexandrov T, Hodgin J, Anderton C . Controlled Humidity Levels for Fine Spatial Detail Information in Enzyme-Assisted -Glycan MALDI MSI. J Am Soc Mass Spectrom. 2022; 33(8):1577-1580. DOI: 10.1021/jasms.2c00120. View

Briggs M, Condina M, Ho Y, Everest-Dass A, Mittal P, Kaur G . MALDI Mass Spectrometry Imaging of Early- and Late-Stage Serous Ovarian Cancer Tissue Reveals Stage-Specific N-Glycans. Proteomics. 2019; 19(21-22):e1800482. DOI: 10.1002/pmic.201800482. View

Buchberger A, DeLaney K, Johnson J, Li L . Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Anal Chem. 2017; 90(1):240-265. PMC: 5959842. DOI: 10.1021/acs.analchem.7b04733. View

Strohalm M, Hassman M, Kosata B, Kodicek M . mMass data miner: an open source alternative for mass spectrometric data analysis. Rapid Commun Mass Spectrom. 2008; 22(6):905-8. DOI: 10.1002/rcm.3444. View

van Hove E, Smith D, Heeren R . A concise review of mass spectrometry imaging. J Chromatogr A. 2010; 1217(25):3946-54. DOI: 10.1016/j.chroma.2010.01.033. View

Reily C, Stewart T, Renfrow M, Novak J . Glycosylation in health and disease. Nat Rev Nephrol. 2019; 15(6):346-366. PMC: 6590709. DOI: 10.1038/s41581-019-0129-4. View

Powers T, Holst S, Wuhrer M, Mehta A, Drake R . Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry. Biomolecules. 2015; 5(4):2554-72. PMC: 4693247. DOI: 10.3390/biom5042554. View

Krenacs L, Krenacs T, Stelkovics E, Raffeld M . Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol. 2009; 588:103-19. PMC: 7604828. DOI: 10.1007/978-1-59745-324-0_14. View

10.

Stambuk T, Klasic M, Zoldos V, Lauc G . N-glycans as functional effectors of genetic and epigenetic disease risk. Mol Aspects Med. 2020; 79:100891. DOI: 10.1016/j.mam.2020.100891. View

11.

Wisztorski M, Franck J, Salzet M, Fournier I . MALDI direct analysis and imaging of frozen versus FFPE tissues: what strategy for which sample?. Methods Mol Biol. 2010; 656:303-22. DOI: 10.1007/978-1-60761-746-4_18. View

12.

Boyaval F, Dalebout H, Van Zeijl R, Wang W, Farina-Sarasqueta A, Lageveen-Kammeijer G . High-Mannose -Glycans as Malignant Progression Markers in Early-Stage Colorectal Cancer. Cancers (Basel). 2022; 14(6). PMC: 8945895. DOI: 10.3390/cancers14061552. View

13.

Varki A . Biological roles of glycans. Glycobiology. 2016; 27(1):3-49. PMC: 5884436. DOI: 10.1093/glycob/cww086. View

14.

Lee Y, Briggs M, Young C, Condina M, Kuliwaba J, Anderson P . Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue. Anal Bioanal Chem. 2022; 414(26):7597-7607. PMC: 9587078. DOI: 10.1007/s00216-022-04289-9. View

15.

Drake R, Powers T, Norris-Caneda K, Mehta A, Angel P . In Situ Imaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or Formalin-Fixed Paraffin-Embedded Tissue. Curr Protoc Protein Sci. 2018; 94(1):e68. DOI: 10.1002/cpps.68. View

16.

West C, Wang M, Herrera H, Liang H, Black A, Angel P . N-Linked Glycan Branching and Fucosylation Are Increased Directly in Hcc Tissue As Determined through in Situ Glycan Imaging. J Proteome Res. 2018; 17(10):3454-3462. PMC: 6784322. DOI: 10.1021/acs.jproteome.8b00323. View

17.

Ohtsubo K, Marth J . Glycosylation in cellular mechanisms of health and disease. Cell. 2006; 126(5):855-67. DOI: 10.1016/j.cell.2006.08.019. View

18.

Hoiem T, Andersen M, Martin-Lorenzo M, Longuespee R, Claes B, Nordborg A . An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue. Proteomics. 2022; 22(10):e2100223. PMC: 9285595. DOI: 10.1002/pmic.202100223. View

19.

Leong A, Haffajee Z . Citraconic anhydride: a new antigen retrieval solution. Pathology. 2009; 42(1):77-81. DOI: 10.3109/00313020903434439. View