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

Abstract

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 high-throughput 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.