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

Our combination of laser micro dissection and mass spectrometry allows for the investigation of spatially resolved changes on the proteomic level in any kind of tissue. In our case, neuromelanin granules. Our protocol allows the proteomic analysis based on limited sample material.

Which is usually a big challenge when working with postmortem brain tissue. Since single cells can be isolated to compare healthy and disease conditions on the proteomic level. This may enhance our understanding of disease mechanisms.

To begin, place the tissue membrane slide in the slide holder on the robo stage with the tissue facing upwards. To acquire an overview scan of the tissue section. Use the scan function, select the area of interest from the top left and the bottom right corners.

Then select scan all ROIs to perform the scan. For the automatic selection of neuromelanin granules in the current field of view. Select field of view analysis.

Then select invert result, and set the threshold for the RGB channels. So that only neuromelanin granules are highlighted in red in the preview window. Now click on okay, to use the adjusted settings for the field of view.

Adjust laser settings using an area of the slide covered by the membrane only. Fill the sample collection tube cap with 50 microliters of ultrapure water. And insert the cap into the collector of the robo mover.

Using the software interface, position the robo mover above the robo stage two to start sample collection. 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 10 objects. When sampling is completed, navigate the robo mover to its starting position and remove the sample collection tube. After spinning the samples by centrification.

Dry the samples for 1.5 hours in a vacuum concentrator. After performing triptic digestion as described in the text. Prepare the sample for HPLC-MS analysis by dissolving 200 to 400 nanograms 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 a low sample amount. Verify identical sample loading by comparing the total ion current.

To begin proteomic raw data analysis. Click load for loading the raw files into the software. Click on set experiment to assign the sample names.

To define group specific parameters. Add the modifications by choosing deamidation NQ, oxidation M, and carbamidomethylation N-term as variable modifications. and then add carbamidomethylation C as fixed modification.

Choose trypsin as a digestion enzyme in the digestion tab. In the label free quantification tab, add the option LFQ and if more than 10 files are to be processed, choose the fast LFQ option to shorten the processing time. Ensuring that all other group specific parameters remain in factory settings.

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 9606 for Homo sapiens. For protein quantification choose unique and razor peptides. Then add the iBAQ option as a measure for protein quantification in the label free quantification tab.

Ensure that all other global parameters remain in factory settings and click on start. After the analysis has been successfully performed retrieve the proteingroups. txt output.

Load the proteingroups. txt file in the analysis software. Add the iBAQ values as main columns and sort all other columns according to their type.

Filter out decoys and contaminants by filtering rows based on the categorical column and filter results based on valid values. Export the output in txt format for further processing. And evaluate the results regarding the research question.

In the present study, 1, 898 protein groups were identified in NMGs and 1, 565 were identified in surrounding SN tissue of which 1, 384 were identified in both tissue areas. Thus 514 and 181 protein groups were exclusively identified in NMGs and SN tissue respectively. In the representative DDA measurements.

Slightly higher iBAQ values in NMGs compared to SN indicated a higher abundance of the protein cytoplasmic dynien-1, heavy chain 1 in NMGs. This observation could be verified in PRM experiments for the peptide ESPEVLLTLDILK. In which a higher abundance in NMGs was detected.

Based on the peak area on MS1 and MS2 level. The most important step is by far the isolation of the NMGs, as the rest of the protocol is based on this step to be properly performed. After LMD procedure, the samples can be utilized and further process for any kind of omics technique such as genomics, transcriptomics, proteomics, or lipidomics.