Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol aimed to describe detailed guidance on the preparation of hard seed sample sections with low water content for MALDI-IMS analysis, maintaining analytes' original distribution and abundance and providing high-quality signal and spatial resolution.

Abstract

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is applied to identify compounds in their native environments. Currently, MALDI-IMS is frequently used in clinical analysis. Still, an excellent perspective exists for better applying this technique to understand chemical compounds' physiological information in plant tissues. However, preparation may be challenging for specific samples from botanical materials, as MALDI-IMS requires thin slices (12-20 µm) for appropriate data acquisition and successful analysis. In this sense, previously, we developed a sample preparation protocol to obtain thin sections of Euterpe oleracea (açaí palm) hard seeds, enabling their molecular mapping by MALDI-IMS.

Here, we show that the developed protocol is suitable for preparing other seeds from the same genus. Briefly, the protocol was based on submerging the seeds in deionized water for 24 h, embedding samples with gelatin, and sectioning them in an acclimatized cryostat. Then, for matrix deposition, an xy motion platform was coupled to an electrospray ionization (ESI) needle spray using a 1:1 (v/v) 2,5-dihydroxybenzoic acid (DHB) and methanol solution with 0.1% trifluoroacetic acid at 30 mg/mL. E. precatoria and E. edulis seed data were processed using software to map their metabolite patterns.

Hexose oligomers were mapped within sample slices to prove the adequacy of the protocol for those samples, as it is known that those seeds contain large amounts of mannan, a polymer of the hexose mannose. As a result, peaks of hexose oligomers, represented by [M + K]+ adducts of (Δ = 162 Da), were identified. Thus, the sample preparation protocol, previously developed tailor-made for E. oleracea seeds, also enabled MALDI-IMS analysis of two other hard palm seeds. In short, the method could constitute a valuable tool for research in the morpho-anatomy and physiology of botanical materials, especially from cut-resistant samples.

Introduction

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is a powerful method that allows two-dimensional biomolecule assignment, provides untargeted investigation of ionizable compounds, and determines their spatial distribution, especially in biological samples1,2. For two decades, this technique has enabled the simultaneous detection and identification of lipids, peptides, carbohydrates, proteins, other metabolites, and synthetic molecules such as therapeutic drugs3,4. MALDI-IMS facilitates chemical analysis in a tissue samp....

Protocol

Euterpe precatoria seeds were kindly donated by the Instituto Nacional de Pesquisas da Amazônia (Manaus, Brazil), and Euterpe edulis seeds were kindly donated by the Silo - Arte e Latitude Rural (Resende, Brazil) after the industrial depulping process. The seeds were maintained in sealed plastic boxes at room temperature.

1. Matrix-assisted laser desorption/ionization-imaging mass spectroscopy (MALDI-IMS)

  1. Seed sectioning protocol
    1. .......

Representative Results

The devised protocol enabled MALDI-IMS analysis of E. precatoria and E. edulis seeds. As a result, we could confirm carbohydrates' molecular weight and degree of polymerization (DP) as a partial structural elucidation. The molecular information provided by the MALDI-IMS analysis (Figure 1 and Figure 2) exhibited peaks representing [M+K]+ adducts of hexose oligomers (Δ = 162 Da) without adding salt to the mat.......

Discussion

Plants are composed of specialized tissues for specific biochemical functions. Therefore, the sample preparation protocol for MALDI-IMS must be designed according to various plant tissues with specific physicochemical properties, as samples must maintain their original analyte distribution and abundance for high-quality signal and spatial resolution8.

Prior to MALDI-IMS analysis, the primary consideration is collecting and storing samples properly. However, in plants, s.......

Acknowledgements

This work was financed by Serrapilheira Institute (Serra-1708-15009), and Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (FAPERJ-JCNE-SEI-260003/004754/2021). Serrapilheira Institute and the National Council for Scientific and Technological Development (CNPq) granted scholarships for Dr. Felipe Lopes Brum and Dr. Gabriel R. Martins (Institutional Capacity Building Program/INT/MCTI). The Coordination for the Improvement of Higher Education Personnel (CAPES) is acknowledged for granting a Master's scholarship for Mr. Davi M. M. C. da Silva. Centro de Espectrometria de Massas de Biomoléculas (CEMBIO-UFRJ) is recognized for ....

Materials

NameCompanyCatalog NumberComments
1 mL Gastight Syringe Model 1001 TLL, PTFE Luer LockHamilton Company81320
2,5-Dihydroxybenzoic acidSigma Aldrich Co, MO, USA149357
APCI needleBruker Daltonik, Bremen, Germany602193
AxiDraw V3 xy motion platformEvil Mad Scientist, CA, USA2510
Carbon double-sided conductive tape
Compass Data Analysis software creation of mass list
Compressed air
copper double-faced adhesive tape3M, USA1182-3/4"X18YD
Cryostat CM 1860 UVLeica  Biosystems, Nussloch, Germany
Diamond Wafering Blade 15 HC
Everhart-Thornley detector
FlexImagingBruker Daltonik, Bremen, Germanyimage acquisition
FTMS ProcessingBruker Daltonik, Bremen, Germanydata calibration
Gelatin from bovine skinSigma Aldrich Co, MO, USAG9391
High Profile Microtome Blades Leica 818Leica  Biosystems, Nussloch, Germany0358 38926
indium tin oxide coated glass slideBruker Daltonik, Bremen, Germany8237001
InkscapeInkscape Project c/o Software Freedom Conservancy, NY, USA
IsoMet 1000 precision cutterBuehler, Illinois, USA
MethanolJ.T.Baker9093-03
Mili-Q water18.2 MΩ.cm
Oil vacuum pump
Optimal Cutting Temperature CompoundFisher HealthCare, Texas, USA4585
Parafilm "M" Sealing FilmAmcorHS234526B
Quanta 450 FEGFEI Co, Hillsboro, OR, USA
SCiLS Lab (Multi-vendor support) MS Software Bruker Daltonik, Bremen, Germany
Software INCA Suite 4.14 VOxford Instruments, Ableton, UK
Solarix 7TBruker Daltonik, Bremen, Germany
Syringe pumpkdScientific, MA, USA78-9100K
Trifluoroacetic acidSigma Aldrich Co, MO, USA302031
X-Max spectrometerOxford Instruments, Ableton, UK

References

  1. Buchberger, A. R., DeLaney, K., Johnson, J., Li, L. Mass spectrometry imaging: a review of emerging advancements and future insights. Analytical Chemistry. 90 (1), 240-265 (2018).
  2. Heeren, R. M. A. MALDITechniques in Mass Spectr....

Explore More Articles

MALDI IMSHard Palm SeedsPlant BiomassSample PreparationImaging Mass SpectrometryMolecular MappingOligosaccharidesBotanical MaterialsSeed DevelopmentGerminationChemical CompoundsTissue Localization

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved