Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Summary

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

Abstract

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

Introduction

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has been employed as an image based technique for two decades^1,2, analyzing a range of biomolecules including: lipids³, peptides^2,4, proteins^2,5, metabolites^6,7, N-glycans⁸, and synthetic molecules such as therapeutic drugs^9,10. The number of publicat....

Protocol

CAUTION: All applicable safety precautions should be followed when performing this procedure, including the use of appropriate personal protective equipment (PPE) (e.g., lab coats, nitrile gloves, safety glasses, etc.)

1. Preparation of Reagents and Equipment

1. Preparation of solutions
   1. To prepare 500 mL of Carnoy's fluid, mix 100% ethanol (EtOH), chloroform, and glacial acetic acid in a 6:3:1 v/v/v ratio in a clean glass Schott bottle.
   2. To make liquid nitrocellulose (NC), dissolve NC membrane (commonly used in western blotting) in neat acetone, by gentle agitation, to a ....

Results

If followed correctly, this protocol produces images that clearly represent the gross morphology of the tissue without any scratches or other deformations (Figure 1). The ideal validation for a correctly performed sample preparation, is the ability to distinguish between different physical structures by changing the molecule being viewed (Figure 2).

A good guide for det.......

Discussion

This protocol was designed to maximize the generation of ionizable molecular species while eliminating delocalization of analytes. The key factors involve using the same overriding principle when applying matrix, digesting the sample, or recrystallizing after sublimation²⁴; namely, that an even deposition of vapor, matrix or otherwise, needs to be created and maintained. Pipetting solvent washes for recrystallization and digestion, evenly underneath the sample, prevents any individual area receivi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryo Microtome	Leica	CM3050	For preparation and section of tissue.
Indium Tin Oxide Microscope slides	Bruker	8237001	For preparation and section of tissue.
Coplin Jars	Sigma Aldrich	S5516	For preparation and section of tissue.
Pressure Cooker	Kambrook	KPR620BSS	For preparation and section of tissue.
Sublimator	Chem Glass	NA	For sublimation procedure. Similar in design to the CG-3038 however it was custom made
Sand bath	NA	NA	For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company
Glass Petri Dish	Sigma Aldrich	CLS70165100	For sublimation procedure.
Vacuum Pump	NA	NA	For sublimation procedure. Sourced as a spare part from an old mass spectrometer
Cold trap	Chem Glass	CG-4510-02	For sublimation procedure.
Hot Plate	John Morris	EW-15956-32.	For sublimation procedure.
Plastic petri dish	Sigma Aldrich	Z717223	For sublimation procedure.
37 °C incubator	NA	NA	For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old
Blotting paper	Sigma Aldrich	P7796	For sublimation procedure.
Nitrocellulose	Sigma Aldrich	N8395	For washing of slides.
Acetone	Sigma Aldrich	650501	For washing of slides.
Xylene	Sigma Aldrich	214736	For washing of slides.
100% EtOH	Sigma Aldrich	1.02428	For washing of slides.
70% EtOH	Sigma Aldrich	NA	For washing of slides. Made in lab from 95% stock ethanol
Chloroform	Sigma Aldrich	C2432	For washing of slides.
Glacial Acetic Acid	Sigma Aldrich	ARK2183	For washing of slides.
Tris HCL pH 8.8	Sigma Aldrich	TRIS-RO	For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8
Milli Q Ultra-Pure Water	Sigma Aldrich	NA	For proteolytic cleavage. Purification performed in house by sartorious water purification system
Ammonium Bircarbonate	Sigma Aldrich	A6141	For proteolytic cleavage.
Trypsin	Sigma Aldrich	T0303	For proteolytic cleavage.
CHCA Matrix	Sigma Aldrich	C2020	For recrystallisation.
Acetonitrile	Sigma Aldrich	1.00029	For recrystallisation.
Trifluoroacetic Acid (TFA)	Sigma Aldrich	302031	For recrystallisation.