Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

The use of human brain tissue was approved by the ethics committee of the Ruhr-University Bochum, Germany (file number 4760-13), according to German regulations and guidelines. This protocol has been applied on commercially obtained substantia nigra pars compacta tissue slices. A graphical overview of the presented protocol is shown in Figure 1.

1. Tissue sectioning

1. Precool the cryostat chamber.
   NOTE: Every tissue requires different cryostat temperatures, which can be found in the respective vendor protocol.
2. Clean the stainless-steel knife with 70% ethanol and install it into the blade holder.
3. Transfer the tissue from the -80 °C freezer to the cryostat using an icebox and let it adjust to the cryostat chamber temperature for 15 min.
4. Unambiguously label membrane slides using a pencil.
   NOTE: PET/PEN membrane slides are required for the LMD-based sample collection. Handle the PET/PEN membrane slides with care as they are extremely fragile.
5. Apply a drop of commercial frozen section medium on the tissue holder. Before it is completely frozen, place the tissue onto the frozen section medium and let it harden, so that the tissue is connected with the tissue holder.
6. Install the tissue holder in the cryostat chamber and adjust its orientation before you start sectioning. Optimal holder orientation depends on the orientation of the tissue.
   NOTE: It may be necessary to trim the tissue until the section plane needed for the slices is reached.
7. Before the tissue area of interest is reached, adjust the cutting setting to the desired tissue thickness.
   NOTE: 5 or 10 µm is the suggested thickness for this protocol as 20 µm thick sections were found to be incompatible with the LMD-based sample collection²².
8. Cut two sections and discard them.
9. Put down the Anti-roll Plate.
10. Cut a section of the tissue, open the Anti-roll Plate carefully, take a membrane slide, and prevent tissue folding while placing the tissue section on the membrane slide.
    NOTE: Storing the membrane slides at room temperature prior to adhesion enables accurate sample attachment. Several sections may be placed on the same membrane slide but tissue overlapping must be prevented.
11. Store tissue sections placed on membrane slides in the cryostat until sectioning is completed.
12. Store the cryosected tissue at -20 °C until further processing or directly proceed with the procedure below. Store the sectioned tissue slides at -80 °C until further use.

2. Laser Microdissection and Pressure Catapulting

NOTE: As neuromelanin granules are visible without any staining due to their black-brownish color, no staining is necessary for this protocol. Nevertheless, different staining procedures can be combined with this protocol if required. Keep in mind that the use of blocking solutions or antibodies will influence the LC-MS/MS analyses.

1. Switch on the MicroBeam system and open the associated software on the computer (see Table of Materials).
2. Place the tissue membrane slide in the SlideHolder on the RoboStage with the tissue facing upwards.
   NOTE: Depending on the LMD device, it may be necessary to place the membrane slide such that the tissue is facing down. In general, sample collection is performed in a temperature-controlled environment to ensure optimal and reproducible conditions.
3. Set the microscope to the desired magnification (50-fold is used here) for the overview scans.
4. Use the Scan function, which can be found in the Navigator window of the software interface, to acquire an overview scan of the tissue section. Search for the top-left corner and the bottom-right corner of the area of interest and select them in the software interface. Then, select Scan all ROIs to perform the scans.
   NOTE: Overview scans are not mandatory, but they enable better orientation in the slide and can be saved for later usage.
5. Adjust the magnification of the microscope for the appropriate tissue, which is 400-fold in the present case of neuromelanin granules.
6. Search for an area with neuromelanin granules. Select Field of View Analysis in the software interface, select Invert Result, and set the threshold for the RGB channels so that only neuromelanin granules are highlighted in red in the preview window. Click on OK to use the adjusted settings for the field of view.
   NOTE: It may occur that smaller objects having a dark color also get selected. To account for that, discard all objects covering an area smaller than 100 µm² before isolating neuromelanin granules. To do this, open the Element List by clicking on the icon in the toolbar, select the slide under consideration and order elements by area. Select those with areas smaller than 100 µm² and delete them.
7. Adjust laser settings using an area of the slide that is covered by the membrane only.
   NOTE: It is suggested to use the Cut Laser Adjustment Wizard and follow the instructions of the software. Required laser settings may differ between different slides. For 5 µm sections with 400-fold magnification, typical settings are 32 energy and 51 focus for cutting, and 28 energy and -1 focus for laser pulse catapulting (LPC).
8. Adjust speed settings for positioning and cutting to ensure proper isolation.
   NOTE: 30% speed was found to be optimal for NMG isolation.
9. Fill the sample collection tube cap with 50 µL ultrapure water and insert the cap into the collector of the RoboMover.
   NOTE: The tube collector used for present experiments can carry one sample collection tube at a time.
10. Position the RoboMover above the RoboStage II using the software interface to start sample collection.
    NOTE: To do this, open the RoboMover window, which displays the collector. Click on the sample collection tube cap displayed in the RoboMover window to move the cap to the working area. Adjust the optimal moving and working height in the RoboMover window. Otherwise, the water in the cap may drop onto the slide or the catapulted objects will not reach the cap.
11. Start the laser. Control energy and focus settings during the laser process and adjust the settings if necessary. Ensure proper isolation and catapulting of the isolated objects into the sample collection tube cap for at least the first ten objects.
    NOTE: Proper isolation and catapulting have to be checked visually. Both should result in a tissue-free area of the size of the pre-selected object in the tissue slice (see Figure 2C,D). Adjust the laser settings if the object stays attached to the tissue slice after cutting and catapulting. For catapulting, the CenterRoboLPC option is found to be well suited for NMG isolation. The catapulting settings can be adjusted for each selected object in the Element List.
12. When sampling is completed, navigate the RoboMover to its starting position. Remove the sample collection tube.
    NOTE: When the number of collected objects is rather low and objects are big enough, sample collection can be ensured by clicking on Cap Check, which will place the sample collection tube cap under the microscope so that the number of objects inside of the water in the cap can be counted (see Figure 2H).
13. Spin down the sample using a centrifuge. Short spins of 5 s with increasing centrifugal force due to acceleration of the centrifuge were found to be sufficient. At this point, store samples at -80 °C, as all samples should be further processed together.
    NOTE: For the comparison of the proteomic profile, the tissue surrounding the NMGs was also isolated after their excision. The isolation of the surrounding tissue was performed at 50-fold magnification.
14. Dry the samples in a vacuum concentrator. 1.5 h were found to be sufficient for 50 µL of water.
15. Solubilize and lyse the tissue in 40 µL formic acid for 20 min (room temperature).
16. Enhance tissue lysis by sonication at 45 kHz (kilohertz) for 5 min in a sonication bath. Fill the sonication bath with ice to prevent tubes from melting. Store the samples at -80 °C until further processing.

3. Tryptic digestion

1. Defreeze samples on ice.
2. Completely dry the samples in a vacuum concentrator.
3. Fill up the sample with 50 µL of a suitable digestion buffer, e.g., 50 mM ammonium bicarbonate.
4. After the addition of 1.25 µL of 200 mM 1,4-dithiothreitol, incubate the samples for 30 min at 60 °C and 300 rpm using a thermomixer and cool them down to room temperature (RT) afterwards.
5. Then, incubate samples at RT for 30 min in the dark after the addition of 1.36 µL 0.55 M iodoacetamide.
6. Add a suitable amount of trypsin to the samples and incubate the samples overnight (~16 h) at 37 °C.
   NOTE: For 1,000,000 µm², 0.1 µg of trypsin was found to be sufficient.
7. Add 2.6 µL of 10% trifluoroacetic acid (TFA) to the samples to stop the digestion (end concentration of 0.5% TFA).
8. Completely dry the samples using a vacuum concentrator. Then, fill samples up to a defined final volume with 0.1% TFA. NMG samples were filled up to 20 µL of which 5 µL were used for one mass spectrometric (MS) experiment.
9. Store the samples at -80 °C until further usage. Determine peptide concentration by amino acid analysis or another suitable quantification method (e.g., Direct Detect).
   ​NOTE: Low sample amounts may not be quantifiable using the mentioned techniques. To ensure identical sample loading, each sample should contain the same amount of isolated tissue and every sample should be treated equally.

4. High-performance liquid chromatography and mass spectrometry

NOTE: The following high-performance liquid chromatography (HPLC) mass spectrometric (MS) analysis are optimized for the specific LC system with a trapping column device and mass spectrometer used here (see Table of Materials). For other LC and MS systems, adaption of parameters is recommended.

1. Using the software Xcalibur, adjust the HPLC settings as follows.
   1. Trap column: Set temperature to 60 °C, flow rate to 30 µL/min, running buffer to 0.1% trifluoroacetic acid.
   2. Analytical C18 reversed-phase column: Set temperature to 60 °C, flow rate to 30 µL/min, running buffer A to 0.1% trifluoroacetic acid, running buffer B to 84% acetonitrile, and gradient to 5%-30% running buffer B over 98 min.
      NOTE: Adaption of the gradient may be inevitable and is strongly recommended when using different tissues or cells. Total gradient time may vary due to sample loading at the beginning of the gradient and sample washing at the end of the gradient. The total gradient in this protocol consists of 7 min sample loading and additional column wash for 15 min resulting in a total gradient time of 120 min.
2. Create a data-dependent acquisition (DDA) method using the XCalibur Instrument Setup, which can be found in the HPLC software roadmap menu.
3. In the Global Parameters tab, define the infusion mode Liquid Chromatography, the Expected LC Peak Width (30 s), and the Default charge state (2).
4. Proceed to the Scan Parameters tab and add the following scans and filters in the order mentioned: MS OT, MIPS, Intensity, Charge State, Dynamic exclusion, and ddMS2 OT HCD.
   NOTE: The detailed parameter settings for each scan and filter can be found in Supplementary Table 1. Optimal MS and DDA settings might vary for the specific mass spectrometer used as well as the sample type and should be, therefore, adapted.
5. Prepare samples by dissolving 200-400 ng of sample peptides in a defined volume of 0.1% TFA in inert mass spectrometric glass vial inlets. If concentration determination is not applicable due to low sample amount, verify identical sample loading by comparing the Total Ion Current (TIC).
   NOTE: To do this, open the resulting file of mass spectrometric measurement in a suitable software, e.g., FreeStyle, and check the chromatogram. Intensities should be comparable for all samples. A representative TIC is shown in Figure 3.
6. Analyze the raw data obtained using a proteomic suitable software, e.g., MaxQuant²³, Progenesis QI for Proteomics, or Proteome Discoverer, and perform a statistical data analysis based on the research question.

5. Analysis of proteomic raw data using MaxQuant

NOTE: A detailed information on MaxQuant parameters is provided in Supplementary Table 2. They are briefly described below.

1. Load raw files into the MaxQuant software in the raw data header by clicking Load.
2. Assign sample names by clicking on Set Experiment.
3. Define group-specific parameters. First, add modifications. Due to sample processing, choose Deamidation (NQ), Oxidation (M), and Carbamidomethylation (N-term) as variable modifications, and add Carbamidomethylation (C) as fixed modification.
4. Choose Trypsin as digestion enzyme in the Digestion tab.
5. Add the label free quantification option LFQ in the Label Free Quantification tab. If more than 10 files are to be processed, choose the Fast LFQ option to shorten the processing time. Add iBAQ option as a measure for protein quantification²⁴.
6. Ensure that all other group-specific parameters remain in factory settings.
7. Proceed to the Global Parameters tab and add the FASTA file derived from uniprot.org in the Sequences tab. Modify the identifier rule accordingly and add the taxonomy ID, in this case, 9606 for homo sapiens.
8. For protein quantification choose Unique and Razor peptides.
9. Ensure that all other global parameters remain in factory settings.
10. Click on Start and retrieve the proteingroups.txt output after MaxQuant analysis for further analysis in Perseus.

6. Statistical analysis using Perseus

1. Load the proteingroups.txt file in Perseus, add the iBAQ values as main columns, and sort all other columns according to their type.
2. Filter out decoys and contaminants by filtering rows based on the categorical column.
3. Filter results based on valid values. In the present case, with only two samples included in the analysis, a minimum number of one valid value was chosen.
4. Export the Perseus output in .txt format for further processing, for example, in Excel, and evaluate the results regarding the research question.

7. Validation of selected proteins

NOTE: Commonly used methods for validation of MS data are, for example, immunological staining or Western Blot. Due to the dark color and the autofluorescence of neuromelanin, immunological staining of proteins inside of neuromelanin granules either with horseradish peroxidase- or fluorophore-conjugated antibodies are not applicable. For Western Blot analysis, very large amounts of post-mortem tissue would be necessary. Therefore, selected proteins are validated by targeted mass spectrometry, and in the present case, parallel reaction monitoring (PRM)-experiments were set up.

1. Select proteins for validation. Choose peptides of these proteins already detected in DDA experiments. Peptides should contain no missed cleavages or modifications to ensure a reliable quantification.
   NOTE: There can be several reasons for the validation of one specific protein, for example, differential abundances in the investigated conditions. For the representative results, cytoplasmic dynein 1 heavy chain 1 has been selected, which was found to be equivalently abundant in NMG and SN samples and could therefore be used as a reference to ensure equal sample loading.
2. Use the selected peptides to set up the first version of a PRM-method using the HPLC software. Keep all chromatography and global parameters settings from the DDA method.
3. Add MS OT and tMS² OT HCD as scan types. Ensure that the settings for MS OT are the same as for the DDA method. Detailed settings for the PRM method can be found in Supplementary Table 1.
4. For tMS² OT HCD, add selected peptides as an inclusion list. Therefore, add the amino acid sequence and the m/z value observed in the DDA measurements. For the first PRM experiment, do not add retention time windows or set t start to 0 and t stop to 120 (for a 120 min gradient).
5. Evaluate the PRM method after the measurement using suitable software, for example, Skyline, and obtain the retention time of the peptides added to the inclusion list. For included peptides, check that comparable peaks are observable for at least three precursor ions in MS1 scans and five fragment ions in MS2 scans with low mass error (±5 ppm).
6. Refine the PRM method, for example, by increasing the resolution for the tMS² OT HCD scan and adding retention time windows to the inclusion list.
   NOTE: Retention time windows of 3 min were found to be well-suited in present experiments (observed retention time in first PRM experiment ± 1.5 min).
7. With the refined PRM-method, perform quantification of peptides and proteins of interest based on the peak area both on MS1 and MS2 levels with suitable software.