The main aim of this procedure is to elucidate the spatial distribution of neuropeptides, specifically opioid peptides directly in thin brain sections using moldy imaging mass spectrometry. This is accomplished by first collecting 12 micrometer thin tissue sections of snap frozen rat brain and tho mounting them onto conductive glass slides. The second step is to apply moldy Matrixx in arrays across the sections using a chemical inkjet printer, and then acquire one mass spectrum from each matrix spot.
Next several peptide peaks are visualized to ensure the overall success of the experiment. This is followed by inspection and processing of the mass spectra export and post-processing of the peptide data and statistical evaluation. The final step is to analyze and validate the peptide data obtained by moldy imaging mass spectrometry.
Ultimately, the results show significant differences in relative opioid peptide levels in different areas of the brain between different treatment groups. The main advantage of this technique over existing methods like radio immuno SAO gel-based approaches is that multi imaging allows for localization of treatment induced changes while maintaining high molecular specificity. This in turn provides us with the possibility to detect like previously not detected truncation of post translation modifications.
Individual new to this method will struggle in the beginning because the technique requires profound sample preparation and data processing. Here the focus will be the quality of the tissue, the matrix, application protocol, data evaluation, and the following validation experiments. It is essential to work quickly and diligently during tissue dissection to avoid proteolytic degradation.
After sacrificing the rat according to approved protocols, immediately remove the brain, transfer it to powdered dry ice, and allow the brain to freeze relatively slowly, store the brain in the minus 80 degrees Celsius freezer. Whole brains can be stored for several years before sectioning without loss of MS signal quality. When ready to begin the procedure, mount the brain on the holder using tissue tech, embedding media, avoid contamination of the part of the brain to be cut as the embedding media interferes with ionization and desorption of peptides.
Cut frozen tissue on a cryostat microtome to 12 micron slices and Thom mount tissue sections on conductive maldi glasss slides. Staining of tissue sections may be performed at this point and the quality of the tissue inspected. This figure shows crestal violet staining of brain tissue flash frozen in liquid nitrogen that caused the tissue to crack and tear.
This tissue would not be appropriate for high resolution moldy imaging mass spectrometry as the matrix will spread and not crystallize evenly dry the sections for 15 minutes under a vacuum and store the slides at minus 80 degrees Celsius until further use tissue section should be analyzed within the shortest possible time after sectioning, even if stored at minus 80 degrees Celsius. First, defrost the sections in a desiccate for one hour, then wash the slides with the sections in 70%ethanol at room temperature for 10 seconds, followed by two washes in 95%Ethanol at room temperature for 10 seconds each. After washing, dry the slides in a desiccate for 10 minutes.
Examine the sections under a microscope to ensure that the tissue sections are of good quality without tissue distortion, micro tears or small cracks that could impair mold EMS quality. Next, prepare fresh matrix solution consisting of 50 milligrams per milliliter, DHB and 50%methanol, 10%150 millimolar ammonium acetate, and 0.3%tri fluoro acetic acid in water. Scan the glass slide holder with the tissue section and align the holder to the respective reference points using the image analysis feature in the chip software.
Then select the appropriate matrix application parameters for both the brain region and the targeted analytes. Next, next, specify the spatial resolution. Now apply the matrix using the optimized protocol on the chemical link Jett printer.
Scan the final matrix spotted sections and save the picture prior to multi-data requisition. This will be used later to cross-reference the matrix deposits with the motor coordinates of the maldi stage. If not used immediately, the matrix spotted sections may be stored in a desiccate under a vacuum until required.
For most experiments, several sections will be analyzed at the same time as seen here. Each slide holder fits two glass slides, and therefore multiple sets of two must be analyzed for all holders. The mold TOF acquisition methods and parameters should be the same, however peptide mass calibrations should be checked for each slide.
Then optimize the acquisition parameters on the tissue sample, including the laser focus and laser intensity. Then, then enter the number of shots needed to achieve an optimal MS signal without elevating the baseline reducing peak resolution. Saturating the detector or ablating matrix from neighboring matrix deposits and select the laser movement.
Estimate the maximum number of shots that can be taken per matrix spot until the only noise is detected here. 600 shots are accumulated from each matrix deposition in 25 shot steps for a total number of 24 steps using a random pattern of movements. Cross-reference the scans of all of the spotted sections with the motor coordinates of the MALDI stage using the flex imaging software.
Then using auto execute batch runner software, perform data acquisition in batch mode. Use data visualization tools such as Flex imaging to visually inspect the Maori IMS images to evaluate peak intensity distributions and determine whether peak intensity distributions are related to tissue features, spotting quality or normalization effects. This example shows the distribution of 10 different peptide peaks in nine different animals.
The total iron current or TIC that is used for normalization is displayed in white. The TIC is the sum of every intensity value in a mass spectrum. The TIC value is very low in animal three, and the peptide images show more noise than specific information.
The brain from animal number six displays an area at the bottom of the section with very high TIC values. This is reflected as abnormally high intensities in that area of peptide images, C and D.This is a potential over normalization effect that can influence data analysis. Over normalization effects can lead to inaccurate results if they are specifically present in only one treatment group.
In this example, the TIC is significantly higher for group C, which can affect individual peak intensities abnormally. For example, the average peak intensity for cytochrome C is higher for group C than group B in the raw data, but after normalization, the peak intensity is now lower. For group C, this can lead to serious misinterpretations of the results.
It is important to always calculate the average TIC for each treatment group before proceeding with extensive data validation. Next, to define the region of interest, it is important to realize that the pro di norphine messenger RNA is translated into the 14 kilodalton pro norphine propeptide. In striate or neurons, the prod norphine is packed into vesicles that are transported through dendrites to either target other striate or neurons, and through axons to the substantial nigra, several millimeters from the cell body.
The vesicles contain enzymes that further process the propeptide into di norphine peptides, such as alpha neo endorphin di norphine A or DI norphine B.Each can activate different opiate receptors upon releasing the target structure, the substantial nigra, the dinorphines are further processed and cleared by endo and exo peptidase. Some of these peptides can only be detected by mass spectrometry as existing antibodies only target the C terminal end of the peptides and not the end terminal. Y-G-G-F-L sequence responsible for opioid receptor activation, the di norphine producing cells reside in the stray AUM and project to the substantial nigra.
Therefore, in the following experiment, both the stray AUM and subs nigra were analyzed by moldy IMS, the spectra from each region of interest. The dorson medial and dorsal lateral stri aum and the medial and lateral nigra were defined in flex analysis software and exported for data analysis. For data reduction, perform peak detection using peak finding tools included in different software.
For example, use peak analysis in origin or MS Peaks in matlab. Export the peak list from all spectra as one single tab delimited text file, determine peak start and stop borders Using software such as the freeway P bin, calculate the area under the curve between peak borders and use these for statistical analysis. MS.Reproducibility between treatment groups can be assessed by calculating the average MS trace and the standard error for each mass value.
In this example, the average spectra for the intact control nigra will used to assess variability across the mass range analyzed. Good reproducibility can be seen as the peak intensities are equal in both control groups and the standard deviation is low. This ensures valid statistical analysis.
If the data sets display a high degree of variation, it is almost always necessary to repeat the Maori IMS experiment, reducing experimental sources of variation as far as possible. Typically, we process as many sections and animals as possible in one single session, sometimes including several people working in shifts. However, in some cases, this is still not enough and other strategies can be employed.
For example, if some spectra consists of noise, these can be by plotting the frequency distribution of the TIC and a cutoff threshold value can be used to eliminate suboptimal data. Another way to evaluate data is to plot all peaks for two animals within the same group against each other. If the peak intensities are equal in both animals, the intensity intensity plot displays a straight narrow line.
If one animal contains low quality spectral data, the plot typically displays asymmetric shifts from the straight line as seen on the right. For peptide sequence identification, perform peptid Dominic analysis on rat brain tissue extracts using multidimensional liquid chromatography hyphenated to high resolution mass spectrometry. Briefly, neuropeptides are extracted from rat brain sections and pre fractionated by means of strong cation chromatography.
The different fractions are then further separated using nano flow reverse phase HPLC. Next, characterize the neuropeptides by means of electro spray tandem mass spectrometry using a hybrid linear ion trap ria transform ion cyclotron resonance instruments finally perform peptide sequence identification by database matching specific antibodies are available for some neuropeptides and immunohistochemistry experiments can validate the peptide identification as well as experimental results. The resolution is higher than for Maori imaging mass spectrometry and additional information can be obtained.
In this example, Dior bee immunoreactive nerve fibers can be seen in the hippocampus, upper left panel and substantial nigra lower left panel, whereas the Maori IMS image on the bottom right reveals the localization at the level of different brain areas. The ultimate proof that a peak mass unequivocally can be assigned to a specific peptide is by performing moldy IMS on peptide knockout animals in the pronin knockout mouse. Several peaks as indicated by the arrows, are missing in the knockout, but not wild type control.
Animals Unrelated peptide peaks such as substance P and P ink display no difference between knockouts and control animals. Di norphine B and alpha neo endorphin peak intensities are significantly increased in the six hydroxy dopamine lesion. Parkinsonian stri AUM of highly dyskinesia animals as indicated by the arrows compared to the low dyskinetic and lesion control group.
Statistical analysis also revealed a significant increase for a peptide mass corresponding to truncated alpha neo endorphin where the end terminal tyrosine was removed. Indeed, visual inspection, the imaging results show high colocalization of the parent di norphine peptides shown in green, and its related DES tyrosine metabolites shown in red yellow signifies regional overlap in the overlay panel to the right. The great potential of this technique is highlighted by the findings concerning selective peptide processing in different brain structures that cannot be detected with other methods.
Here, specific DES tyrosine metabolites shown in red, co localized with the parent peptide shown in green and were only observed in the striatum, but not the substantial nigra of dyskinetic rats. In the sub nigra image analysis revealed that the degradation product lu and kelin arch shown in red was inversely localized to di norphine B shown in green, indicating that during dyskinesia, the DI norphine B was locally released in the lateral subs, nigra, and then metabolized to lu and careful in AR One set up. A single experiment using mold imaging can be performed in one day or two for sample preparation and data acquisition.
This then followed by another day or two for data evaluation. Depending on sample size, the validation experiments would require another day or two hands-on experiments in the lab if performed properly. It's important to take particular care in the following steps.
Tissue dissection and freezing has to be done quickly in order to avoid pep degradation, washing and matrix application protocol have to be optimized for each experiment. Data evaluation and statistical analysis have to be evaluated carefully in order to avoid false positives due to over normalization effects or poor peak detections.
