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Abstract: FR-OR099

Visualizing the N-Linked Glycome within Human Kidney Biopsies Using Mass Spectrometry Imaging

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Anderton, Christopher R., Pacific Northwest National Laboratory, Richland, Washington, United States
  • Velickovic, Dusan, Pacific Northwest National Laboratory, Richland, Washington, United States
  • Zhang, Guanshi, University of Texas Health San Antonio, San Antonio, Texas, United States
  • Bhattacharjee, Arunima, Pacific Northwest National Laboratory, Richland, Washington, United States
  • Pasa-Tolic, Ljiljana, Pacific Northwest National Laboratory, Richland, Washington, United States
  • Alexandrov, Theodore, European Molecular Biology Laboratory, Heidelberg, Germany
  • Sharma, Kumar, University of Texas Health San Antonio, San Antonio, Texas, United States

Group or Team Name

  • for the KMPM Consortium
Background

The current study is a part of the NIDDK Kidney Precision Medicine Project (KPMP). The observed alterations in protein glycosylation attributed to disease development has stemmed major interest in studying glycans (e.g., higher HbA1c in type 1 diabetes is associated with changes in the serum N-glycome). Currently, a limited number of methods are available for interrogating clinical tissue samples for variations in protein glycosylation. An emerging approach is on-tissue enzymatic N-glycan releasing (OtER) followed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which positionally conserves the location of N-glycan moieties and allows their composition to be registered to histological information. This method can be performed on archival, formalin-fixed paraffin-embedded (FFPE), clinical sample tissue.

Methods

We optimized the OtER MALDI-MSI workflow on FFPE preserved health human biopsy tissue provided by the KPMP. Tissue was sectioned and mounted on conductive glass slides, deparaffinized by washing with xylenes, rehydrated with EtOH:H2O solutions, and then antigen retrieved (citraconic buffer). On-tissue application of Peptide-N-Glycosidase F was applied with a robotic sprayer, followed by incubation in a humidified environment (40 oC). Finally, matrix (cyanohydroxycinnamic acid) was applied to samples and MALDI was performed.

Results

Several high mannose, hybrid, and paucinomannose N-glycans were spatially detected in the human kidney tissue, and many showed co-localization with different histological features, as revealed through pre- and post-MALDI autofluorescence and H&E images. MALDI performed using the high mass resolution 15T Fourier transform ion cyclotron resonance MS gave confident matches of N-glycans in the ChEBI database and with those previously reported. We were able to separate isomeric N-glycans using pre-mass analysis ion mobility separation in our MALDI-MSI method.

Conclusion

This highlights the value of using this workflow for mapping the N-glycome, where the location of species can be registered to different anatomical compartments and cell types within the human kidney. We anticipate this method can provide the ability to distinguish diseased from healthy kidney biopsies, by identifying aberrant N-glycosylation patterns.

Funding

  • NIDDK Support