Application & Background
2,5-dihydroxybenzoic acid (DHB) is a matrix suitable for imaging metabolites. DHB can easily be dissolved in 50:50 Water:Methanol (0.1% Trifluoroacetic Acid) solution. When imaging metabolites DHB can be sprayed directly onto tissue sections with no pre-treatment, but may show interfering peaks in the low mass region of the mass spectra. The data presented here were obtained as part of a MALDI MS imaging experiment whose purpose was to detect metabolites present in the root nodules of the Medicago truncatula – Sinorhizobium meliloti symbiosis during nitrogen fixation.
Figure 1. Methylene blue stained Medicago truncatula root nodule (Image courtesy of Dr. Jean-Michel Ané lab in the Department of Agronomy at UW-Madison.)
Experimental
experimental design
Root nodules were dissected out of the plant, embedded in gelatin (100 mg/ml in deionized water), and frozen on dry ice. Cryosections (14 microns) of snap frozen root nodule tissue was mounted on ITO-coated glass slides.
Matrix Application using the HTX TM Sprayer
Tissue sections were then sprayed with DHB matrix (40mg/ ml, Methanol 50%, TFA 0.1%) using the HTX TM-Sprayer and the following conditions:
Table 1. Spraying parameters for matrix deposition using the HTX TM Sprayer.
MALDI Imaging
Spectra were collected across the entire tissue area using the ultrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Billerica, MA, USA) analyzer equipped with a 2 kHz, FlatTop smartbeam-II™ Nd:YAG laser in reflectron positive mode over a mass range of m/z 80 to 1000. A total of 500 laser shots were accumulated and averaged from each laser spot, using the “minimum” laser spot diameter setting and a raster width of 50μm. Calibration was performed externally using DHB cluster peaks in the mass range of m/z 100 to 750.
results
Figure 2. A) Overlaid images of m/z 342.44, 603.85, and 673.83 which show spatial differentiation in different parts of the root nodule. B) Distribution of m/z 342.44 localized to the nitrogen fixation zone region. C) Distribution of m/z 603.85 localized to the outer nodule region. D) Distribution of m/z 673.83 localized to the root region.
Figure 3. Reference Optical Image
Figure 4. Optical image of serial tissue sections before (left) and after (right) MALDI- MSI. The area of tissue that was imaged is outlined in red on the right.
Figure 5. Zoomed in image of matrix crystals showing crystal size and coverage.
Figure 6. Mass spectra for the three distinct regions of the root nodule tissue: outer nodule, nitrogen fixation zone, and root. Metabolites with m/z 80-1000 were imaged. The inlay zooms in on the region m/z 80-300 which has the greatest abundance of different metabolites. The calculated [M+H]+ for FMRFamide is 599.3, which was added to the matrix as an internal calibrant.
Figure 7. Medicago truncatula- intact plant before dissection (Image courtesy of Dr. Jean-Michel Ané in the Department of Agronomy at UW-Madison.)
The tissue images and MS data presented in this note were provided by Erin Gemperline (Department of Chemistry) and Dr. Lingjun Li (Department of Chemistry and School of Pharmacy), University of Wisconsin-Madison, Madison, WI, USA