研究目的
Investigating the enhancement of on-tissue chemical derivatization by laser-assisted tissue transfer for MALDI MS imaging of small endogenous metabolites in tissues.
研究成果
The LATT system significantly improves the performance of on-tissue chemical derivatization for MALDI MSI of endogenous small molecules by reducing ion suppression effects and increasing the number of derivatized metabolites detected with higher signal intensities. It also minimizes analyte delocalization and maximizes the spatially resolving power of MALDI MSI.
研究不足
The technique requires specific laser-absorbing substances to facilitate tissue transfer, and the flatness and the thickness of tissue film could be affected by a few factors such as the stability of the laser energy level and the levelness of the machine.
1：Experimental Design and Method Selection:
The LATT system was assembled with a 450 nm semiconductor blue laser, an electrospray emitter, and a 2D high-resolution translation stage. Derivatization reagent and matrix solution were delivered through the electrospray emitter.
2：Sample Selection and Data Sources:
Brain tissues were harvested from male 8-week-old Sprague-Dawley rats. The brains were surgically dissected and snap-frozen immediately in dry ice prior to store at -80 °C.
3：List of Experimental Equipment and Materials:
2,5-dihydroxybenzoic acid (DHB), quercetin, 9-aminoacridine (9-AA), 1,5-diaminonapthalene (DAN), 4-hydroxy-3-methoxycinnamaldehyde (CA), and trifluoroacetic acid (TFA), GirT reagent, a-cyano-4-hydroxycinnamic acid (CHCA), theanine, valine, γ-aminobutyric acid (GABA), dopamine, dexamethasone and bovine serum albumin (BSA).
4：Experimental Procedures and Operational Workflow:
The mixture of the matrix and derivatization reagent was deposited onto tissue sections by the electrospray emitter. The tissue sections on the donor slide were placed on the top of the acceptor slide in a face-to-face manner prior to being mounted on the 2D translation stage. The laser beam was focused at the tissue layer, generated strongly localized heat to force a small volume of tissue to be ablated from the donor slide onto the acceptor slide to form a thin-layer film.
5：Data Analysis Methods:
MALDI MSI data was read and analyzed using flexAnalysis 3.4 and flexImaging 4.1. The comparisons of peak intensities from different tissue regions were performed by extracting intensity data from defined regions of interest (ROI) by MSiReader v1.00.
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trifluoroacetic acid
TFA
Sigma-Aldrich
Used as an additive in the derivatization reagent solution to improve derivatization efficiency.
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2,5-dihydroxybenzoic acid
DHB
Sigma-Aldrich
Used as a matrix in MALDI MSI to facilitate the ionization of analytes.
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9-aminoacridine
9-AA
Sigma-Aldrich
Used as a matrix in MALDI MSI to facilitate the ionization of analytes.
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1,5-diaminonapthalene
DAN
Sigma-Aldrich
Used as a matrix in MALDI MSI to facilitate the ionization of analytes.
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4-hydroxy-3-methoxycinnamaldehyde
CA
Sigma-Aldrich
Used as a derivatization reagent for amino-specific derivatization in MALDI MSI.
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quercetin
Sigma-Aldrich
Used as a matrix in MALDI MSI to facilitate the ionization of analytes.
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Girard reagent T
GirT
Shanghai Yuanye Bio-Technology
Used as a derivatization reagent for steroids in MALDI MSI.
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a-cyano-4-hydroxycinnamic acid
CHCA
Shanghai Yuanye Bio-Technology
Used as a matrix in MALDI MSI to facilitate the ionization of analytes.
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theanine
Shanghai Yuanye Bio-Technology
Used as a standard compound to assess the performance of the LATT system.
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valine
Shanghai Yuanye Bio-Technology
Used as a standard compound to assess the performance of the LATT system.
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γ-aminobutyric acid
GABA
Shanghai Yuanye Bio-Technology
Used as a standard compound to assess the performance of the LATT system.
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dopamine
Shanghai Yuanye Bio-Technology
Used as a standard compound to assess the performance of the LATT system.
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dexamethasone
Shanghai Yuanye Bio-Technology
Used as a standard compound to assess the performance of the LATT system.
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bovine serum albumin
BSA
Shanghai Yuanye Bio-Technology
Used to validate the potential laser-induced dissociation of peptides or proteins.
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Peptide calibration standard II
Bruker Daltonics
Used to validate the potential laser-induced dissociation of peptides or proteins.
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HPLC grade methanol
MeOH
Merck
Used as a solvent for preparing matrix and derivatization reagent solutions.
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Ultrapure deionized water
Millipore
Used as a solvent for preparing matrix and derivatization reagent solutions.
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