This study involves brain tissue samples from human participants, reviewed and approved by TMH and IITB IEC – (IITB-IEC/2018/019). The participants provided their informed and written consent to participate in this study.

1 Protein extraction from brain tissue

1. Weigh around 50 mg of brain tissue and wash the tissue with 300 µL of 1x phosphate buffer saline (PBS) using a micropipette.
   NOTE: This step is performed to remove any blood on the external surface of the tissue and must be repeated if necessary. It is advisable to remove as much blood from tissue as possible as it interferes with downstream protein estimation and processing.
2. Following washes with PBS, add 300 µL of lysis buffer (Buffer A) to the tube containing the tissue. Buffer A contains 8 M urea, 50 mM Tris pH 8.0, 75 mM NaCl, 1 mM MgCl₂ and Protease inhibitor cocktail (as per manufacturer's instructions).
   NOTE: Protein yield varies depending on several factors ranging from the conditions in which the samples are stored, the amount of starting material and the efficiency of handling the samples while processing. Reduce the volume of buffer A proportionately when working with amounts of tissue lower than 50 mg.
3. Lyse the tissue using a probe sonicator while keeping the tube on an ice bath. Use the following parameters for sonication: 40% amplitude, 5 seconds on and off cycle for 2:30 min.
4. Continue with tissue homogenization using a bead beater to completely lyse the tissue.
   NOTE: This step should be performed at medium speed for 90 seconds followed by incubation on ice for 3-5 minutes.
5. Centrifuge the contents of the tube at 6,000 x g for 15 min at 4 °C. Transfer the supernatant into a fresh tube and mix homogeneously.
   ​NOTE: Make aliquots of the sample and store at -80°C until further use.

2 Protein quantification and quality check

1. Quantify the samples prior to digestion using a standard graph made with known concentrations of BSA.
   ​NOTE: Ensure that the assay being used for protein estimation is compatible with the buffer used for making the protein lysate. Check the quality of the protein lysate by running an SDS-PAGE gel.

3 Protein digestion

1. Take 50 µg of protein in a micro-centrifuge tube and reduce the proteins by adding Tris 2-carboxyethyl phosphine (TCEP) such that the final concentration is 20 mM. Incubate the contents at 37 °C for 1 h.
2. Following incubation, alkylate the reduced proteins by adding iodoacetamide (IAA) to the tube such that its final concentration is 40 mM. Incubate the tube in dark for 30 minutes.
   NOTE: Prepare IAA freshly right before its addition to the tube.
3. Add buffer B to the tube containing the reduced and alkylated proteins such that the final concentration of urea in the tube is less than 1 M.
   NOTE: Buffer B is composed of 25 mM Tris (pH 8.0) and 1 mM CaCl₂ and is used for diluting the samples. Upon dilution, ensure that the contents of the tube have a pH of 8 for optimum protein digestion after addition of trypsin.
4. Add trypsin in a 1:30 enzyme to protein ratio and incubate the tubes overnight at 37 °C with constant shaking.
5. Following digestion, concentrate the digested product in a vacuum concentrator. At this step, the peptides can either be reconstituted and desalted or stored at -80 °C for future use.

4 Desalting and peptide quantification

NOTE: Desalting or peptide clean-up is essential before loading the samples for LC-MS/MS. Salts and other contaminants in the sample can clog the columns and cause damage to the instrument as well. The process can be performed using commercially available C18 stage-tips or columns.

1. Activate the stage tip with 20 µL of 50% acetonitrile (ACN) in 0.1% formic acid (FA). Centrifuge the contents at 1,000 x g for 1 minute and discard the flow through.
   NOTE: Conditions for centrifugation are the same till the end of the procedure.
2. Add 20 µL of 100% ACN in 0.1% FA and centrifuge the contents as in step 4.1.
   NOTE: Activation steps can be repeated a couple of times.
3. Following activation, pass 20 µL of reconstituted peptide sample through the stage-tip and centrifuge as performed in step 4.1.
   NOTE: Do not discard the flow through in this step.
4. Repeat step 4.3 with the flow through at least 5 times to ensure maximal peptide binding to the stage-tip.
5. Pass 20 µL of 0.1% FA through the stage-tip and discard the flow through.
   NOTE: Repeat this step two more times for better results.
6. Elute the bound peptides in a fresh microfuge tube by passing increasing concentrations of ACN, i.e., 40%, 60% and 80%, respectively.
7. Dry the peptides in a vacuum concentrator and proceed for peptide quantification.
8. Reconstitute the dried peptides in 0.1% FA and quantify using the Scopes method⁵.

5 Transition list preparation of finalized targets

NOTE: A transition refers to the pair of precursors (Q1) to product (Q3) m/z values in an MRM experiment. A peptide can have one to many transitions, with the same Q1 value but different Q3 values. A triple quadrupole mass spectrometer requires information of the transitions for the peptides and their products to be detected. Hence, before starting a targeted experiment, a transition list needs to be prepared. This can be done using the online repository of SRMAtlas⁶ (https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/GetTransitions) or an open source software called Skyline⁷ (https://skyline.ms/project/home/software/Skyline/begin.view).

1. Download the recent human proteome FASTA file from UniProt (https://www.uniprot.org/) and create a background proteome file by inserting it in Skyline. In peptide settings, go to the Background Proteome dropdown and click on Add. Feed in the FASTA sequence file of the human proteome. Make sure this database is selected and the allowed missed cleavage value is set to 0 before proceeding to the next step.
2. Now, under the Filter tab in Peptide Settings delimit the length of accepted peptides from 8 to 25 amino acids.
3. In Transition Settings, under Filter tab set Precursor Charges as 2,3, set Ion Charges as 1 and set Ion Types to y. Product ions can be selected depending on the user's choice. Select N-terminal to proline for special ions and leave all other parameters as default.
   NOTE: The peptide and transition settings can vary according to the experiment.
4. Insert the peptides or proteins of interest by clicking on Edit and moving to Insert. To insert proteins, copy their accession IDs and to insert specific peptides, copy the peptide sequences.The software maps the accession IDs to the background proteome and the transition list is created based on the peptide and transition settings.
5. Export the transition list. Ensure that in the dropdown for Instrument Type, the right instrument is selected. For the optimization experiments, one may choose to split the transition lists into smaller numbers by setting the desired number of transitions per file in Skyline. This will ensure that the instrument is not overwhelmed to screen too many transitions in a single run. The number of transitions needs to be further optimized to get a single method – this we mention henceforth under the method refinement section.

6 LC parameters

1. Use a binary solvent system with the aqueous solvent containing 0.1% FA (Solvent A) and the organic solvent containing 80% ACN (Solvent B).
2. Set the column temperature to 45 °C.
3. Set an LC gradient of 10 min with a 450 µL/min flowrate (as shown in Table S1).

7 MS parameters

NOTE: The explained assay has been developed and optimized for TSQ Altis Triple Quadrupole Mass Spectrometer.

1. Optimize runs parameters like Q1 and Q3 resolution, dwell time and cycle time – one parameter at a time. We find that 0.7 resolution for Q1 and Q3 works best. The cycle time or dwell time might need to be tweaked according to the number of transitions and average peak width of the peptides being monitored.
2. Use the MS parameters in Table S2 and Table S3. The total method duration is 10 min.
   ​NOTE: The parameters remain same for all the runs during and after method refinement, unless mentioned otherwise. For a fresh experiment, there may be a need to change certain parameters depending on the type of the sample and sample processing steps.

8 Run sequence and Instrument QC

1. Prepare a mixture of water: methanol: isopropanol: acetonitrile in 1:1:1:1 ratio. Use this mixture as a blank.
2. Prepare peptides for any standard sample that can be used to consistently monitor the performance of the instrument. This will be used as a QC standard. We detect peptides from BSA digests that have been optimized and give good and consistent response over several days (Figure 2).
3. To run samples, the sequence should start with a couple of blanks, followed by the QC standard and clinical samples. Always ensure there is one blank between two consecutive samples.
4. For the ease of comparison, ensure equal amounts and volumes of each sample are injected every time.

9 Method refinement

1. Analyze the preliminary data obtained from the pooled samples using Skyline. Look for the right peak, transitions, and parameters such as peak shape and intensity to select the best results. Save the file as a new Skyline project.
2. Use a library to find the best matching peak and consequently the best possible transition list is advised. A library is a set of MS/MS peaks available from literature or in-house experiments.
3. Export a fresh transition list from the newly saved Skyline project and use this transition list to make a fresh method. Acquire data from the new method created and repeat the process of refining the transitions using Skyline.
4. Once the list of peptides and transitions is finalized following data analysis using Skyline, fine tune the method by trying out different permutations of cycle time and dwell time.
   ​NOTE: The use of heavy labelled synthetic peptides and indexed retention time (iRT) peptides makes method refinement easy^{8, 9}. It is hence advisable that fine tuning of the method be performed using these peptides.
5. Using the retention time information from the refinement experiments, create a final method that is scheduled (having a defined acquisition window).
6. Prepare samples for individual subjects. Data will be acquired for these samples using the final scheduled method.
7. Once the data is acquired, further downstream analysis and group-wise comparison can be performed by importing the raw files to Skyline.