Sequential Detection of Biomolecules in Formalin-fixed, Paraffin-embedded Samples with Mass Spectrometry Imaging

Summary

Here, we present a Mass Spectrometry Imaging protocol for sequential metabolite, N-linked glycan, and tryptic peptide detection in formalin-fixed, paraffin-embedded tissue samples.

Abstract

Tissues are complex cellular environments, made up of a vast array of cell types and biomolecules all interacting with each other to carry out the functions of the organ. Traditionally, techniques for the analysis of biomolecules such as metabolites, glycans, and proteins involved the homogenization of tissue, destroying all spatial information. These traditional methods inhibited the complete understanding of complex intra- and extracellular molecular interactions. Mass Spectrometry Imaging (MSI), on the other hand, not only preserves the spatial information of biomolecules in tissue but also allows for multiple classes of analytes to be detected from the same tissue section through sequential analysis at a near-single-cell resolution. This enables us to derive a more complete picture of molecular interactions across different classes of biomolecules. The protocol presented here outlines the steps for performing mass spectrometry imaging of metabolites, N-linked glycans, and tryptic peptides sequentially from the same tissue section at a 20 µm resolution. Conscientious consideration of the order in which the classes of analytes are imaged, along with careful handling of the sections to ensure integrity, allows for multiple high-quality images to be collected from the same section. These data can subsequently be integrated with other spatial omics data (transcriptomics, immunohistochemistry, etc.) collected from serial sections, where the same cell can be analyzed in these adjacent sections.

Introduction

Mass Spectrometry Imaging (MSI) is a powerful technique that allows for the detection and visualization of hundreds to thousands of biomolecules from thin tissue sections, without the need for a priori knowledge of the exact molecules present in the tissue, making it an excellent tool for biomarker discovery¹. While several types of MSI are used by different labs, including Desorption ElectroSpray Ionization (DESI)^2,3, Infrared Matrix Assisted Laser Desorption ElectroSpray Ionization (IR-MALDESI)⁴, and Secondary Ion Mass Spectrometry (SIMS)

Protocol

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissues collected from previously untreated patients undergoing primary cytoreductive surgery for high-grade serous ovarian carcinoma. All clinical data were obtained from the ovarian cancer repository of the Department of Gynecologic Oncology and Reproductive Medicine under protocols approved by the University of Texas MD Anderson's Institutional Review Board. Written informed consent from the patients was obtained by front desk personnel, and the studies were conducted in accordance with recognized ethical guidelines.

1. Metabolite imaging sample preparation

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Results

The completion of this protocol should result in three robust MSI datasets from the same section of tissue. In the visualization of the full spectrum of the metabolite data, the spectrum will be heavily dominated by matrix peaks at m/z 157 and 315. This is normal for FFPE tissue. Many metabolite and fatty acid signals will be observed by zooming in on the m/z ranges <155 and between 250 and 300. Figure 3 shows examples of the full spectrum and zoomed ranges highlighting the complex metabo.......

Discussion

The collection of sequential MSI data from a single tissue section requires careful attention to detail. There are a few steps that are absolutely crucial to achieving high-quality data. First, care should be taken if more than one slide is prepared at the same time. When placing the slides into, or moving between Coplin jars, be careful not to place two slides into the same position in the jar. This will result in inadequate solvent exposure of the tissue surface, or one slide can scrape the tissue off the other slide. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,5-diaminonaphthalene	Fisher Scientific	D010125G	MALDI Matrix for metabolite imaging
Acetonitrile	Fisher Scientific	A955-4	LC-MS grade solvent used for matrix preparation
α-cyano-4-hydroxycinnamic acid	Sigma-Aldrich	70990-1G-F	MALDI matrix for glycan and peptide imaging
Ammonium Bicarbonate	Fisher Scientific	A643-500	Buffer for enzymes
Ammonium Phosphate	Sigma-Aldrich	467782-50G	Additive to reduce matrix clusters during imaging
Decloaking Chamber NxGen	Biocare Medical	N/A	Used for antigen retrieval of tissue
Ethanol	Fisher Scientific	04-355-223	LC-MS grade solvent used for matrix preparation and staining
M5 Robotic Reagent Sprayer	HTX Imaging	N/A	Used for application of enzymes and matrices to tissue
Methanol	Fisher Scientific	A456-4	LC-MS grade solvent for making red phosphorus suspension
MS Grade Trypsin	Fisher Scientific	PI90058	Enzyme for protein digestion
MTP Slide Adapter II	Bruker Daltonics	8235380	Adapter to insert micrscope slides into mass spectrometer
NanoZoomer SQ Digital Slide Scanner	Hamamatsu Corp	N/A	Used for generating digital microscopy images of stained tissue
Perfection V600 Flatbed Scanner	Epson	N/A	Used for generating optical image of the slide for MSI data collection
Petri-seal	Fisher Scientific	50-212-518	For sealing petri dish during enzymatic digestion
PNGaseF	Bulldog Bio	NZPP550LY	Enzyme for cleavage of N-linked glycans from proteins
Red Phosphorus	Sigma-Aldrich	04004-250G	MALDI calibrant for both positive and negative ion mode
SCiLS Lab (2025b)	Bruker Daltonics	N/A	Software for MSI data visualization
timsTOF fleX QTOF Mass Spectrometer	Bruker Daltonics	N/A	Used for mass spectrometry data collection
Trifluoracetic acid	Fisher Scientific	85183	Matrix additive to decrease pH for positive ion mode imaging
Tris Base	Fisher Scientific	BP152-500	Buffer for antigen retrieval
Water	Fisher Scientific	W64	LC-MS grade solvent used for matrices, enzymes, and staining
WypAll X60	Fisher Scientific	19-413-113	Absorbent wipe for humidified enzyme incubation
Xylene	Fisher Scientific	X3P-1GAL	Clearing agent for staining