Effective Sample Preparations in Imaging Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) can be used to visualize the distribution of biomolecules (proteins, peptides, metabolites) and drugs on tissue surfaces. In MALDI-IMS, sample preparation is crucial for successful results. A variety of conditions, such as tissue sampling methods, tissue thickness and matrix application procedure can have an impact on the results. In this review, we summarize each sample preparation step in an orderly sequence with practical examples. In addition, we discuss the importance of the organic solvent used in the matrix solution. The composition of the organic solvent used in the matrix solution is critical for achieving a high sensitivity in this procedure.

Keywords: adenylates; anti-cancer agents; focused-microwave irradiation; imaging mass spectrometry; sample preparation.

Fig. 1. Workflow for IMS and possible parameters in each step.

Fig. 2. Comparison of labile metabolites distributions using different sampling methods.¹⁴⁾ The FMW-treatment successfully suppressed these changes and resulted in high quality distribution images. (FMW; focused microwave irradiation, ISF; in-situ freezing, and PEF; post-euthanized freezing method)

Fig. 3. MALDI-MS protein profiles obtained from sections of mouse brain. (a) Optical image of a mouse brain section mounted on a gold-coated slide glass and sprayed with a matrix solution of sinapinic acid (12.5 mg/mL in 50% ACN/0.1% TFA). The mouse brain sections were prepared at thicknesses of 2, 5, 10, 20, 30, 40 μm. All mass spectra were acquired from the cerebral cortex represented as a white circle in inset. (b) Examples of mouse brain imaging acquired from slices prepared at different thicknesses. High molecular weight proteins were difficult to observe in the thicker sections.¹⁵⁾

Fig. 4. Tissue shrinkage due to organic solvent and matrix homogeneity. Comparison of tissue surface (a) before and (b) after spraying matrix solution without matrix deposition. Bottom pictures are enlarged views of the tissue surface. (c) Tissue surface following the two-step method.²⁰⁾

Fig. 5. Comparison of ion intensity between the dry-coating method and the two-step application method. (a) olaparib detection in the dry-coating method; (b) erlotinib detection in the dry-coating method; (c) olaparib detection in the two-step method; (d) erlotinib detection in the two-step method.

Fig. 6. Time-lapse observation of crystal formation on (a) two-step application and (b) conventional method (direct droplet). Crystals observed with scanning electron microscope in (c) two-step application and (d) conventional method.²¹⁾

Fig. 7. Comparison of ion intensity in product ion spectra of olaparib on α-CHCA-coated ITO glass slides. The 0.1 μL of premixed olaparib and matrix solution were spotted onto the glass slides. The type of acid used in the matrix solution affected the ion intensity; (a) 0.1% formic acid (FA) and (b) 0.1% trifluoroacetic acid (TFA). For olaparib detection, the peak intensity of olaparib in 0.1% FA was higher by three times than that in 0.1% TFA.

Fig. 8. Comparison of ion intensity using different solvent composition on an olaparib dosed mouse liver section. (a) 50% ACN/0.1% FA; (b) 50% EtOH/0.1% FA; (c) 50% MeOH/0.1% FA. Solvent in the matrix solution affected ion intensity due to its extraction efficiency and solubility. The intensity for 50% ACN/0.1%FA was approximately higher by 5 times than that for 50% MeOH/0.1% FA.

Fig. 9. Comparison of organic solvent concentration in the matrix solution. (a) 50% ACN/0.1% FA; (b) 30% ACN/10% IPA/0.1% FA; (c) 40% ACN/10% IPA/0.1% FA; (d) 50% ACN/10% IPA/0.1% FA. The addition of 10% IPA slightly improved the ion intensity in olaparib detection. At higher concentrations, the solvent probably evaporated before sufficient extraction could be achieved.