MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

Thomas W Powers,Huiyuan Tang,Dean A Troyer,Richard R Drake,Brian B Haab,Anand S Mehta,Benjamin A Neely,Yuan Shao,Surinder K Batra

doi:10.1371/journal.pone.0106255

Abstract

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

Highlights

Tissues obtained from surgeries or diagnostic procedures are most commonly preserved in formalin-fixed paraffin-embedded (FFPE) tissue blocks
Mouse kidney tissues were fixed in formalin and used as an initial model system to develop MALDI-IMS glycan imaging workflows for FFPE tissues
Multiple N-linked glycans can be directly profiled from FFPE tissue blocks and tissue microarray (TMA) while maintaining intact architecture

Summary

Introduction

Tissues obtained from surgeries or diagnostic procedures are most commonly preserved in formalin-fixed paraffin-embedded (FFPE) tissue blocks. When combined with clinical outcomes, FFPE tissues are a rich source of samples for biomarker discovery and validation in retrospective studies. Incorporation of multiple FFPE tumor tissue cores in a tissue microarray (TMA) format has proven to be effective for immunohistochemistry analysis of potential biomarker candidates [5], and TMAs are increasingly being used for validation of alterations in protein expression associated with emerging genetic mutation phenotypes and transcriptional profiling studies [6,7]. When correlated with associated clinical outcomes, this provides a powerful method for biomarker discovery and validation while minimizing reagent use and assuring that each core in the TMA is treated under identical conditions

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Results

Conclusion