This procedure spatially localizes multiple endogenous lipids within a bovine lens. Using the technique of matrix assisted laser DESORPTION ionization, mass spectrometric imaging. First, prepare a thin bovine lens tissue section and apply a multi matrixx, then acquire a maldi mass spectral dataset.
Process the dataset into a visual representation of the detected lipids on the lens tissue section. Together these results can determine the location and relative abundance of the lipids within a bovine lens and produce a visual representation of this distribution Compared to other methods such as lipid extraction followed by LCMS analysis, maldi mass spectrometry imaging allows for the visualization of multiple lipids simultaneously without losing their spatial localization. Remove the flash frozen tissue specimens from minus 80 degrees Celsius storage.
Remove the capsule from the surface of the lens to fix a whole bovine calf lens section. Place one or two drops of water on the tissue cutting stage of a cryostat. Quickly place the lens in the water before it solidifies when the cryostat has equilibrated to optimal temperature.
Cut the tissue equatorially into sections of 20 micron thickness. Discard the first tissue sections and only use slices that are close to or add the equatorial plane for imaging ocular lens tissue. Add 1.5 microliters of formic acid to pre-wet the surface of an ITO coated glass slide.
Then carefully transfer the tissue sections to the slide inside the cryostat. Next, lyophilize the slides for 15 minutes Before maldi matrix application. Using a correction fluid pen, add three teaching marks on the non-conductive surface of the ITO coated glass slide.
Now take an optical image of the tissue slide using a flatbed scanner and save it in an appropriate format such as TIFF or jpeg. First, cover the edges on the front surface of the ITO coated glass slides with tape so that the matrix does not coat the edges of the slide. Prepare the matrix to be used in an appropriate solvent.
Then apply matrix solutions to the surfaces of the tissue sections. Coat the glass slide by using 20 cycles of matrix coating. Alternatively, a pneumatically assisted airbrush sprayer can be used to apply the matrices if a solvent is incompatible with the electronic matrix sprayer.
For the mass calibration solution, dilute the ES tuning mix standard solution by a factor of one to 200 in 60%Isopropanol introduced two microliters per minute of the diluted ES tuning mix solution into the dual mode electro spray ionization ion source on the instrument for four a transform ion cyclotron resonance ms. Operate the F-T-I-C-R instrument in the positive ion ESI mode with broadband detection and a data acquisition size of 1024 kilobytes per second. Typically set the ESI parameters of 3, 900 volts capillary electro spray voltage.
A spray shield voltage of 3, 600 volts nebulizer nitrogen gas flow at two liters per minute. Dry nitrogen gas flow at four liters per minute and temperature of 200 degrees Celsius. Also set the skimmer one voltage to 15 volts.
Time of flight to 0.01 seconds Collision argon gas flow to 0.4 liters per second source ion accumulation time to 0.1 second and the collision cell ion accumulation time to 0.2 seconds. Now tune the F-T-I-C-R operation parameters in order to maximize the analytical sensitivity over the mass range from mass to charge ratio 200 to 1400 while maintaining good time domain free induction decay signals. Then acquire the ESI mass spectra and calibrate the instrument using the reference masses of the standard compounds in the E es tuning mix solution.
To tune the instrument for MALDI operation, dissolve several one microliter aliquots of a mixed turine and INE standard solution in the matrix solution at a concentration of one Micromolar each and spot these solutions directly onto one of the sample tissue sections. Place the slide into a tissue slide adapter and load the adapter from the MALDI side into the dual ESI maldi ion source. Next, optimize the appropriate MALDI operating parameters for the laser power and the number of laser shots for MALDI signal accumulation for each mass scan.
After tuning, calibrating and optimizing the instrument for maldi MSI experiments, align the physical location of a tissue section to be imaged with its recorded optical image within the imaging software. Align the three correction fluid markings using a three point triangulation method. Then begin a simultaneous ESI MALDI operation so that each mass spectrum contains the reference mass peaks of the E ES tuning mix solution for post-acquisition internal mass calibration.
First attenuate the ESI signal by decreasing the capillary voltage until the MALDI signals dominate the spectra, while the ESI caliber signals are still high enough for internal mass calibration. Next, set up an automated rasing method for laser of radiation. Using a random spot analysis.
Define the tissue regions to be imaged and set the appropriate laser RA step size. Calibrate the MALDI mass spectra using internal calibration for the initial comparison, and to select peaks for MSMS de isotope and select the mono isotopic peaks using a customized VBA script and export the resulting mono isotopic peak lists. Input the measured mass to charge ratio values into the Melin nine and or the HMDB metabolome databases for mass matching with the library entries.
Next, generate maldi images for all of the lipid entities detected across the entire tissue section using imaging software with a mass filter width of one part per million at the peak apex. Once images have been generated for all mass to charge ratio values that match database entries generate images for all other peaks as well to look for unique distribution patterns that can be investigated later for some specific tissues, such as bovine lens. Extensive tearing of the tissue is often observed when direct thaw mounting is used.
Preco of the IT OAS slide with ethanol or formic acid helps to maintain the integrity of the tissue sections during tissue mounting. Both the choice of matrix and the selection of solvent are important factors influencing the quality of the MALDI spectra. These examples compare a spectrum produced with an efficient matrix solvent with a poor choice of matrix solvent for diol.
Once the set of mass spectra from a Maldi MSI experiment has been acquired, the image for each of the detected ions can be generated. With each pixel representing a laser irradiation spot from the surface of a tissue section. The relative concentrations of the analytes can be detailed in the different portions of the tissue section.
In most experiments, the data is normalized the total ion current within each spectrum. Without this normalization areas with better analyte matrix, COC crystallization could cause stronger signals for the analytes, and this would skew the data. Tissue preparation can also dramatically change the image that is generated.
If the sample is too wet, then the analytes will delocalize on the tissue and much of the spatial information will be lost. This demonstration of a tissue imaging experiment on ocular lens used diol as a multi Matrixx to determine the spatial distribution of lipids by F-T-I-C-R-M-S. Imaging of lipids in different types of tissue can be accomplished using diol or another matrix in a similar way.
