Chemical Decrosslinking-Based Peptide Characterization of Formaldehyde-Fixed Rat Pancreas Using Fluorescence-Guided Single-Cell Mass Spectrometry

Approaches for the characterization of proteins/ peptides in single cells of formaldehyde-fixed (FF) tissues via mass spectrometry (MS) are still under development. The lack of a general method for selectively eliminating formaldehyde-induced crosslinking is a major challenge. A workflow is shown for the highthroughput peptide profiling of single cells isolated from FF tissues, here the rodent pancreas, which possesses multiple peptide hormones from the islets of Langerhans. The heat treatment is enhanced by a collagen-selective multistep thermal process assisting efficient isolation of islets from the FF pancreas and, subsequently, their dissociation into single islet cells. Hydroxylamine-based chemical decrosslinking helped restore intact peptide signals from individual isolated cells. Subsequently, an acetone/ glycerol-assisted cell dispersion was optimized for spatially resolved cell deposition onto glass slides, while a glycerol solution maintained the hydrated state of the cells. This sample preparation procedure allowed peptide profiling in FF single cells by fluorescence-guided matrix-assisted laser desorption ionization MS. Here, 2594 single islet cells were analyzed and 28 peptides were detected, including insulin C-peptides and glucagon. T-distributed stochastic neighbor embedding (t-SNE) data visualization demonstrated that cells cluster based on cell-specific pancreatic peptide hormones. This workflow expands the sample availability for single-cell MS characterization to a wide range of formaldehyde-treated tissue specimens stored in biobanks.

Automated summary

Approaches for protein/peptide characterization in single cells of formaldehyde-fixed tissues via mass spectrometry are still being developed. The lack of a general method to eliminate formaldehyde-induced crosslinking is a major challenge. A workflow was established for high-throughput peptide profiling of single cells from formaldehyde-fixed pancreatic tissues, enabling the analysis of 2594 single islet cells and detection of 28 peptides, including insulin C-peptides and glucagon. This workflow expands the sample availability for single-cell MS characterization to a wide range of formaldehyde-fixed tissues stored in biobanks.