The main contribution of our paper is that we have explored a precise and fine landscape of amyloid beta pathology of the Alzheimer's disease brain with the imaging technology of the MALDI imaging mass spectrometry. We achieved technological advancements by generating a special protocol with formic acid pre-treatment of the tissue slides. Following MALDI-IMS, segmentation maps are generated and calculations are performed to discover novel marker proteins or peptides colocalized with plaque and subarachnoid vasculature.

Obtain human cortical specimens for IMS from brains that were removed, processed, and stored at minus 80 degrees Celsius within eight hours post-mortem. Take the brain specimen from the occipital cortex of AD patients and age-matched controls. To cut the tissue sections on a cryostat, first place conductive indium tin oxide or ITO-coated microscope glass slides inside the cryostat.

Warm the autopsy brain specimen from minus 80 degrees Celsius to minus 22 degrees Celsius inside the cryostat. Attach a new disposable blade to the cryostat for every experiment. Always try to use a clean part of the blade.

Put the frozen autopsy brains on the stage along with a small amount of optimal cutting temperature compound. For IMS and immunohistochemistry, cut five to six sections from each tissue sample. When the blade is just starting to cut the tissue, turn the wheel and face the block until all of the tissue is exposed.

If there is a small streak or tear across the section, wait in the cryostat until the temperature adjustment automatically fixes it. Count a few seconds before opening the anti-roll with a tissue underneath. Immediately place the tissue slice on the ITO-coated side of the glass slide.

Thaw the tissue slice by putting a finger underneath the slide on the non-ITO-coated side. The tissue will stick to the slide. Ensure that the tissue is as flat as possible with no wrinkles.

To rinse the tissue sections, immerse the samples in 40 to 100 milliliters of 70%ethanol in a glass staining jar for 30 seconds to remove endogenous lipids and inorganic salts. Wash the samples using the washing sequence listed in the text protocol. Then, dry the samples in a vacuum for 30 minutes.

Now, treat the tissue sections with a formic acid vapor for a better ionization of the amyloid beta proteins from autopsy brain tissue. To do so, prepare the oven at 60 degrees Celsius and an incubation glass dish with five milliliters of 100%formic acid. Keep the air humidity in the incubation glass dish at saturation level.

Place the tissue slides in the incubation glass dish while avoid submersion in the formic acid and treat for six minutes. Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope. Perform this step at room temperature.

The alignment of the optical image of the samples is necessary when the sample target is placed inside the instrument. Usually, it will not be possible to recognize the tissue section underneath the matrix layer. To correlate the optical images with the samples, make guide marks that are visible both in the optical image and underneath the matrix layer in the camera optic.

The easiest way is to spot at least three correction fluid marks around the sample before taking the optical image. To spray the matrix with an ultrasonic sprayer, remove the tissue to be sprayed from the desiccator and place it in the chamber. Make sure the tissue is not covering the sensor window.

Start the preparation by pushing the Start button. Usually, prep time is around 90 minutes. The preparation will be regulated automatically via the monitoring of the matrix layer thickness and wetness.

Alternatively, to spray the matrix solution on the tissue surface with an automatic sprayer, use a solvent pump system set at 10 psi and 0.15 milliliters per minute to deliver the matrix solution. A constant flow of heated sheath gas will be delivered conjointly with the matrix solution spray. Perform high-throughput and high-spatial-resolution imaging experiments with MALDI-IMS.

For mass spectrometry measurement, define the tissue areas using the MALDI control software and data analysis software. Acquire spectra in a positive linear mode with a mass to charge ratio range of 2, 000 to 20, 000 and a spatial resolution of 20 and 100 microns. To make the calibration standard, dissolve the peptide calibration standard and the protein calibration standard in a one to four ratio with CHCA and TA30 solution and then dilute it 10 times.

Place one microliter of calibration standard on the slide at four different locations. Using molecular histology software, overlay multiple signal images to find the spatial correlation of various signals such as different amyloid beta peptides colocalizing in senile plaques and arterial walls. The cerebral amyloid angiopathy or CAA phenotypes of patient number three were most prominent in this study.

MALDI-IMS of this patient's brain tissue clearly visualized that amyloid beta 1-42 and amyloid beta 1-43 were preferentially deposited as senile plaques in the cerebral parenchyma. By contrast, shorter amyloid betas such as amyloid beta 1-36 to 1-41 were preferentially deposited on the leptomeningeal vascular areas. There were no significant signals in the non-pathological control.

Distributions of amyloid beta 1-40 and amyloid beta 1-42 were further validated with immunohistochemistry using adjacent frozen sections of the tissues. The anti-amyloid beta 1-40 antibody labeled CAA and revealed amyloid beta 1-40 is preferentially deposited in leptomeningeal blood vessels, which is clear contrast to the distribution of amyloid beta 1-42 in the cerebral parenchyma as senile plaques. Shown here is a segmentation map obtained with a bisecting k-means analysis applied to the same section from patient number three.

This clustering method successfully identified plaque-like structures in the parenchyma and vascular structures in the subarachnoid space. It is interesting to find a small circular area in the parenchyma which is detected just around a small arteriole in the parenchyma as well as in the subarachnoid space. This is verified by single ion images of these individual amyloid beta peptides.

There is a limit to trace a broad range of amyloid betas simultaneously even if using the specific antibodies. Here, we show the distribution of amyloid beta in human brains by using the cutting-edge MALDI imaging mass spectrometry method. We believe this contribution makes breakthrough in Alzheimer disease research because this report offers the characterization of the full set of amyloid beta proteins in human autopsied brains.

The most impressive finding is that only one single amino acid alteration at the C terminal of amyloid beta protein makes a drastic change in their distribution. The tissue preparation steps are critical to obtain an effective ionization of aggregated proteins in human brain tissue. Homogeneous cocrystallization of the analyte with matrixes crucial for high sensitivity and artifact-free imaging.

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Amyloid Beta

MALDI Imaging Mass Spectrometry