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.

Figure 1.

Schematic of image-guided single cell MALDI MS analysis of formaldehyde-fixed pancreas showing the major processing and cell deposition steps.

Figure 2.

Fluorescence-guided single cell MALDI MS analysis. (a) Schematic of microMS-assisted fluorescence image-guided single-cell localization to determine each cell’s relative coordinates. (b) Brightfield image of dissociated pancreatic islet cells on an ITO-coated glass slide. (c) Fluorescence image before and after (d) microMS-based image filtering and cell localization. (e) Mass spectra showing peptide hormones in chemically restored alpha-cell (red line) and beta-cell (black line) isolated from FF pancreatic tissue.

Figure 3.

MALDI-MS analysis of 2594 islet cells isolated from FF pancreas. (a) peptide peaks selected from average mass spectrum of the mass spectra acquired from 2594 cells. (b) peptide signals used in t-SNE analysis. (c) t-SNE plots of data points for six peptide hormones plotted on t-SNE of entire data set. Alpha-cell expresses glicentin-related polypeptide, m/z 3379.0; glucagon, m/z 3482.5 and beta-cell express insulin-2 C-peptide, m/z 3160.9; insulin-1 C-peptide, m/z 3259.0; insulin-1 B-chain, m/z 3441.1; insulin, m/z 5800.9.