Skip to main content
SpringerLink
Log in

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AVS:

Aortic valve stenosis

CS:

Chondroitin sulfate

ECM:

Extracellular matrix

HRAM:

High-resolution accurate mass

IMS:

Imaging mass spectrometry

GAG:

Glycosaminoglycan

MALDI:

Matrix-assisted laser desorption ionization

PTM:

Post-translational modification

TIMS-TOF:

Trapped ion mobility time of flight

References

  1. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci. 2010;123:4195–200.

    Article  CAS  Google Scholar 

  2. Schaefer L, Schaefer RM. Proteoglycans: from structural compounds to signaling molecules. Cell Tissue Res. 2010;339:237–46.

    Article  CAS  Google Scholar 

  3. Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem. 2009;78:929–58.

    Article  CAS  Google Scholar 

  4. Gordon MK, Hahn RA. Collagens. Cell Tissue Res. 2010;339:247–57.

    Article  CAS  Google Scholar 

  5. Sethi MK, Downs M, Zaia J. Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis. Mol Omics. 2020;16(4):364–76.

    Article  CAS  Google Scholar 

  6. Ruhaak LR, Xu G, Li Q, Goonatilleke E, Lebrilla CB. Mass spectrometry approaches to glycomic and glycoproteomic analyses. Chem Rev. 2018;118:7886–930.

    Article  CAS  Google Scholar 

  7. Raghunathan R, Sethi MK, Zaia J. On-slide tissue digestion for mass spectrometry based glycomic and proteomic profiling. MethodsX. 2019;6:2329–47.

    Article  Google Scholar 

  8. Norris JL, Caprioli RM. Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research. Chem Rev. 2013;113:2309–42.

    Article  CAS  Google Scholar 

  9. Gessela M, Spraggins JM, Voziyanb P, Hudsonb BG, Caprioli RM. Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix. J Mass Spectrom. 2015;50:1288–93.

    Article  Google Scholar 

  10. Angel PM, Schwamborn K, Comte-Walters S, Clift C, Ball LE, Mehta AS, et al. Extracellular matrix imaging of breast tissue pathologies by MALDI imaging mass spectrometry. Proteomics Clin Appl. 2018;1700152:1700152.

    Google Scholar 

  11. Angel PM, Comte-Walters S, Ball LE, Talbot K, Mehta AS, Brockbank KGMM, et al. Mapping extracellular matrix proteins in formalin-fixed, paraffin-embedded tissues by MALDI imaging mass spectrometry. J Proteome Res. 2018;17:635–46.

    Article  CAS  Google Scholar 

  12. Powers TW, Neely BA, Shao Y, Tang H, Troyer DA, Mehta AS, et al. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays. PLoS One. 2014;9:e106255.

    Article  Google Scholar 

  13. Heijs B, Holst S, Briaire-De Bruijn IH, Van Pelt GW, De Ru AH, Van Veelen PA, et al. Multimodal mass spectrometry imaging of N-glycans and proteins from the same tissue section. Anal Chem. 2016;88:7745–53.

    Article  CAS  Google Scholar 

  14. Angel PM, Mehta A, Norris-Caneda K, Drake RR. MALDI imaging mass spectrometry of N-glycans and tryptic peptides from the same formalin-fixed, paraffin-embedded tissue section. Methods Mol Biol. 2018;1788:225–41.

    Article  CAS  Google Scholar 

  15. Clift C, Mehta A, Drake RR, Angel PM. Multiplexed imaging mass spectrometry of histological staining, N-glycan and extracellular matrix from one tissue section: a tool for fibrosis research. Methods Mol Biol. 2020;xx:xxx.

  16. Aichler M, Kunzke T, Buck A, Sun N, Ackermann M, Jonigk D, et al. Molecular similarities and differences from human pulmonary fibrosis and corresponding mouse model: MALDI imaging mass spectrometry in comparative medicine. Lab Investig. 2018;98:141–9.

    Article  CAS  Google Scholar 

  17. Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res. 2008;7:1650–9.

    Article  CAS  Google Scholar 

  18. Yamauchi M, Sricholpech M. Lysine post-translational modifications of collagen. Essays Biochem. 2012;52:113–33.

    Article  CAS  Google Scholar 

  19. Zhang Y, Fonslow BR, Shan B, Baek MC, Yates JR. Protein analysis by shotgun/bottom-up proteomics. Chem Rev. 2013;113:2343–94.

    Article  CAS  Google Scholar 

  20. Zhao RR, Ackers-Johnson M, Stenzig J, Chen C, Ding T, Zhou Y, et al. Targeting chondroitin sulfate glycosaminoglycans to treat cardiac fibrosis in pathological remodeling. Circulation. 2018;137:2497–513.

    Article  CAS  Google Scholar 

  21. Westergren-Thorsson G, Hedstrom U, Nybom A, Tykesson E, Ahrman E, Hornfelt M, et al. Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol. 2017;83:27–38.

    Article  CAS  Google Scholar 

  22. Guo S, Xue C, Li G, Zhao X, Wang Y, Xu J. Serum levels of glycosaminoglycans and chondroitin sulfate/hyaluronic acid disaccharides as diagnostic markers for liver diseases. J Carbohydr Chem. 2015;34:55–69.

    Article  Google Scholar 

  23. Zaia J. Glycosaminoglycan glycomics using mass spectrometry. Mol Cell Proteomics. 2013;12:885–92.

    Article  CAS  Google Scholar 

  24. Turiák L, Tóth G, Ozohanics O, Révész Á, Ács A, Vékey K, et al. Sensitive method for glycosaminoglycan analysis of tissue sections. J Chromatogr A. 2018;1544:41–8.

    Article  Google Scholar 

  25. Tacha D, Teixeira M. History and overview of antigen retrieval: methodologies and critical aspects. J Histotechnol. 2002;25:237–42.

    Article  CAS  Google Scholar 

  26. Magangane PS, Khumalo NP, Adeola HA. The effect of antigen retrieval buffers on MALDI mass spectrometry imaging of peptide profiles in skin FFPE tissue. J Interdiscip Hist. 2018;6:26–32.

    Article  Google Scholar 

  27. Klein JA, Meng L, Zaia J. Deep sequencing of complex proteoglycans: a novel strategy for high coverage and sitespecific identification of glycosaminoglycanlinked peptides. Mol Cell Proteomics. 2018;17:1578–90.

    Article  CAS  Google Scholar 

  28. Smirnov IP, Zhu X, Taylor T, Huang Y, Ross P, Papayanopoulos IA, et al. Suppression of α-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry. Anal Chem. 2004;76:2958–65.

    Article  CAS  Google Scholar 

  29. Wei J, Wu J, Tang Y, Ridgeway ME, Park MA, Costello CE, et al. Characterization and quantification of highly sulfated glycosaminoglycan isomers by gated-trapped ion mobility spectrometry negative electron transfer dissociation MS/MS. Anal Chem. 2019;91:2994–3001.

    Article  CAS  Google Scholar 

  30. Miller RL, Guimond SE, Schwörer R, Zubkova OV, Tyler PC, Xu Y, et al. Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides. Nat Commun. 2020;11:1481.

    Article  CAS  Google Scholar 

  31. Stephens EH, Saltarrelli JG, Baggett LS, Nandi I, Kuo JJ, Davis AR, et al. Differential proteoglycan and hyaluronan distribution in calcified aortic valves. Cardiovasc Pathol. 2011;20:334–42.

    Article  CAS  Google Scholar 

  32. Fornieri C, Baccarani-Contri M, Quaglino D, Pasquali-Ronchetti I. Lysyl oxidase activity and elastinglycosaminoglycan interactions in growing chick and rat aortas. J Cell Biol. 1987;105:1463–9.

    Article  CAS  Google Scholar 

  33. Itabashi T, Harata S, Endo M, Takagaki K, Yukawa M, Ueyama K, et al. Interaction between proteoglycans and α-elastin in construction of extracellular matrix of human yellow ligament. Connect Tissue Res. 2005;46:67–73.

    Article  CAS  Google Scholar 

  34. Reinboth B, Hanssen E, Cleary EG, Gibson MA. Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1. J Biol Chem. 2002;277:3950–7.

    Article  CAS  Google Scholar 

  35. Duarte AS, Correia A, Esteves AC. Bacterial collagenases – a review. Crit Rev Microbiol. 2016;42:106–26.

    Article  Google Scholar 

  36. Shibata S, Midura RJ, Hascall VC. Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1. J Biol Chem. 1992;267:6548–55.

    Article  CAS  Google Scholar 

  37. Yao X, Freas A, Ramirez J, Demirev PA, Fenselau C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal Chem. 2001;73:2836–42.

    Article  CAS  Google Scholar 

  38. West C. Determination of N-linked glycosylation changes in hepatocellular carcinoma and the associated glycoproteins for enhanced biomarker discovery and therapeutic targets. 2020. Medical University of South Carolina, Charleston, SC, USA.

  39. West CA, Liang H, Drake RR, Mehta AS. New enzymatic approach to distinguish Fucosylation isomers of N-linked glycans in tissues using MALDI imaging mass spectrometry. J Proteome Res. 2020;19:2989–96.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Dr. Shannon Cornett for his help with ion mobility experiments.

Funding

Funding for this work was provided by the American Heart Association (16GRNT31380005) to PMA with additional support by the NIH/NIGMS (P20 GM103542) and National Center for Advancing Translational Sciences (UL1 TR000445), which supported initial studies for the project. CLC supported by HL007260 (NIH/NHLBI). Support to RRD was provided NCI/IMAT (1R21CA207779). RRD and ASM were supported by the South Carolina Centers of Economic Excellence SmartState program.

Author information

Authors and Affiliations

  1. Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA

    Cassandra L. Clift, Richard R. Drake, Anand Mehta & Peggi M. Angel

Authors
  1. Cassandra L. Clift
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard R. Drake
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anand Mehta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peggi M. Angel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Cassandra Clift and Dr. Peggi Angel. The first draft of the manuscript was written by Cassandra Clift and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Peggi M. Angel.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Code availability

Not applicable.

Additional information

Published in the topical collection Mass Spectrometry Imaging 2.0 with guest editors Shane R. Ellis and Tiffany Porta Siegel.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

(PDF 0.99 mb).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Clift, C.L., Drake, R.R., Mehta, A. et al. Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections. Anal Bioanal Chem 413, 2709–2719 (2021). https://doi.org/10.1007/s00216-020-03047-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-03047-z

Keywords

Search

Navigation