We acknowledge MHRD-UAY Project (UCHHATAR AVISHKAR YOJANA), project #34_IITB to SS and MASSFIITB Facility at IIT Bombay supported by the Department of Biotechnology (BT/PR13114/INF/22/206/2015) to carry out all MS-related experiments.
We extend our special thanks to Mr. Rishabh Yadav for making and editing of the entire video and Mr. Nishant Nerurkar for his work in editing the audio.

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&#160;Protein extraction from brain tissue
<ol>
	<li>Weigh around 50 mg of brain tissue and wash the tissue with 300 &#181;L 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.</li>
	<li>Following washes with PBS, add 300 &#181;L 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&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge the contents of the tube at 6,000 x g for 15 min at 4 &#176;C. Transfer the supernatant into a fresh tube and mix homogeneously. 
	&#8203;NOTE: Make aliquots of the sample and store at -80&#176;C until further use.</li>
</ol>
2 Protein quantification and quality check
<ol>
	<li>Quantify the samples prior to digestion using a standard graph made with known concentrations of BSA. 
	&#8203;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.</li>
</ol>
3 Protein digestion
<ol>
	<li>Take 50 &#181;g 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 &#176;C for 1 h.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add trypsin in a 1:30 enzyme to protein ratio and incubate the tubes overnight at 37 &#176;C with constant shaking.</li>
	<li>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 &#176;C for future use.</li>
</ol>
4 Desalting and peptide quantification
NOTE: Desalting or peptide clean-up is essential before loading the samples for&#160;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.
<ol>
	<li>Activate the stage tip with 20 &#181;L 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.</li>
	<li>Add 20 &#181;L 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.</li>
	<li>Following activation, pass 20 &#181;L 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.</li>
	<li>Repeat step 4.3 with the flow through at least 5 times to ensure maximal peptide binding to the stage-tip.</li>
	<li>Pass 20 &#181;L of 0.1% FA through the stage-tip and discard the flow through. 
	NOTE: Repeat this step two more times for better results.</li>
	<li>Elute the bound peptides in a fresh microfuge tube by passing increasing concentrations of ACN, i.e., 40%, 60% and 80%, respectively.</li>
	<li>Dry the peptides in a vacuum concentrator and proceed for peptide quantification.</li>
	<li>Reconstitute the dried peptides in 0.1% FA and quantify using the Scopes method5.</li>
</ol>
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)&#160;or an open source software called Skyline7 (https://skyline.ms/project/home/software/Skyline/begin.view).
<ol>
	<li>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.</li>
	<li>Now, under the Filter tab in Peptide Settings delimit the length of accepted peptides from 8 to 25 amino acids.</li>
	<li>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&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6 LC parameters
<ol>
	<li>Use a binary solvent system with the aqueous solvent containing 0.1% FA (Solvent A) and the organic solvent containing 80% ACN (Solvent B).</li>
	<li>Set the column temperature to 45 &#176;C.</li>
	<li>Set an LC gradient of 10 min with a 450 &#181;L/min flowrate (as shown in Table S1).</li>
</ol>
7 MS parameters
NOTE: The explained assay has been developed and optimized for TSQ Altis Triple Quadrupole Mass Spectrometer.
<ol>
	<li>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.</li>
	<li>Use the MS parameters in Table S2 and Table S3. The total method duration is 10 min. 
	&#8203;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.</li>
</ol>
8 Run sequence and Instrument QC
<ol>
	<li>Prepare a mixture of water: methanol: isopropanol: acetonitrile in 1:1:1:1 ratio. Use this mixture as a blank.</li>
	<li>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).</li>
	<li>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.</li>
	<li>For the ease of comparison, ensure equal amounts and volumes of each sample are injected every time.</li>
</ol>
9 Method refinement
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: The use of heavy labelled synthetic peptides and indexed retention time (iRT) peptides makes method refinement easy8, 9. It is hence advisable that fine tuning of the method be performed using these peptides.</li>
	<li>Using the retention time information from the refinement experiments, create a final method that is scheduled (having a defined acquisition window).</li>
	<li>Prepare samples for individual subjects. Data will be acquired for these samples using the final scheduled method.</li>
	<li>Once the data is acquired, further downstream analysis and group-wise comparison can be performed by importing the raw files to Skyline.</li>
</ol>

<ol><li>Picotti, P., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selected+reaction+monitoring-based+proteomics%3A+Workflows%2C+potential%2C+pitfalls+and+future+directions%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Selected reaction monitoring-based proteomics: Workflows, potential, pitfalls and future directions.</a> Nature Methods. , (2012).</li>
<li>Carr, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+peptide+measurements+in+biology+and+medicine%3A+Best+practices+for+mass+spectrometry-based+assay+development+using+a+fit-for-purpose+approach%5BTitle%5D)+AND+%22Molecular+and+Cellular+Proteomics%22%5BJournal%5D)">Targeted peptide measurements in biology and medicine: Best practices for mass spectrometry-based assay development using a fit-for-purpose approach.</a> Molecular and Cellular Proteomics. 13 (3), 907-917 (2014).</li>
<li>Pitt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+and+applications+of+liquid+chromatography-mass+spectrometry+in+clinical+biochemistry%5BTitle%5D)+AND+%22The+Clinical+biochemist+Reviews%22%5BJournal%5D)">Principles and applications of liquid chromatography-mass spectrometry in clinical biochemistry.</a> The Clinical biochemist Reviews. 30 (1), 19-34 (2009).</li>
<li>Hosp, F., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Primer+on+Concepts+and+Applications+of+Proteomics+in+Neuroscience%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A Primer on Concepts and Applications of Proteomics in Neuroscience.</a> Neuron. 96 (3), 558-571 (2017).</li>
<li>Scopes, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+protein+by+spectrophotometry+at+205+nm%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Measurement of protein by spectrophotometry at 205 nm.</a> Analytical Biochemistry. , (1974).</li>
<li>Kusebauch, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+SRMAtlas%3A+A+Resource+of+Targeted+Assays+to+Quantify+the+Complete+Human+Proteome%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.</a> Cell. , (2016).</li>
<li>MacLean, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skyline%3A+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li>
<li>Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absolute+quantification+of+proteins+and+phosphoproteins+from+cell+lysates+by+tandem+MS%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2003).</li>
<li>Escher, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+iRT%2C+a+normalized+retention+time+for+more+targeted+measurement+of+peptides%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Using iRT, a normalized retention time for more targeted measurement of peptides.</a> Proteomics. , (2012).</li>
<li>Gillette, M. A., Carr, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+peptides+and+proteins+in+biomedicine+by+targeted+mass+spectrometry%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry.</a> Nature Methods. 10 (1), 28-34 (2013).</li>
<li>Whiteaker, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+targeted+proteomics-based+pipeline+for+verification+of+biomarkers+in+plasma%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A targeted proteomics-based pipeline for verification of biomarkers in plasma.</a> Nature Biotechnology. , (2011).</li>
<li>Hüttenhain, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducible+quantification+of+cancer-associated+proteins+in+body+fluids+using+targeted+proteomics%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.</a> Science Translational Medicine. 4 (142), 94(2012).</li>
<li>Mermelekas, G., Vlahou, A., Zoidakis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SRM%2FMRM+targeted+proteomics+as+a+tool+for+biomarker+validation+and+absolute+quantification+in+human+urine%5BTitle%5D)+AND+%22Expert+Review+of+Molecular+Diagnostics%22%5BJournal%5D)">SRM/MRM targeted proteomics as a tool for biomarker validation and absolute quantification in human urine.</a> Expert Review of Molecular Diagnostics. 15 (11), 1441-1454 (2015).</li>
<li>Koldamova, R. P., Lefterov, I. M., Lefterova, M. I., Lazo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apolipoprotein+A-I+directly+interacts+with+amyloid+precursor+protein+and+inhibits+A%CE%B2+aggregation+and+toxicity%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits Aβ aggregation and toxicity.</a> Biochemistry. , (2001).</li>
<li>Jiang, S. X., Slinn, J., Aylsworth, A., Hou, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vimentin+participates+in+microglia+activation+and+neurotoxicity+in+cerebral+ischemia%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Vimentin participates in microglia activation and neurotoxicity in cerebral ischemia.</a> Journal of Neurochemistry. , (2012).</li>
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The authors received support from Thermofisher for the publication fee.

Techniques like Immunohistochemistry and Western blotting were considered as the gold standards for validation of protein targets for many years.&#160;These methods find use even today with minor modifications in the protocol and little dependence on technology making them very cumbersome and tedious. Besides this, they also involve the use of expensive antibodies which do not always show the same specificity across batches and require a great deal of expertise. Additionally, only a small fraction of proteins identified ...

During the last decade, rapid developments in mass spectrometry (MS) coupled with increased understanding of chromatography techniques have greatly helped in advancement of MS-based proteomics. Molecular biology-based techniques such as western blotting and immunohistochemistry have long suffered from reproducibility issues, slow turnaround time, inter-observer variability and their inability to accurately quantify proteins, to name a few. To this end, the superior sensitivity of high-throughput proteomics approaches continues to offer molecular biologists an alternate and more reliable tool in their quest to better understand the roles of proteins in cells. However, shotgun proteomics approaches (Data dependent Acquisition or DDA) often fail to detect low abundant proteins in complex tissues besides being heavily reliant on the sensitivity and resolution of the instrument. Over the last couple of years, labs around the world have been developing techniques like Data Independent Acquisition (DIA) that require increased computing power and reliable software that can handle these highly complex datasets. However, these techniques are still a work in progress and not very user friendly. Targeted MS-based proteomics approaches provide a perfect balance between the high throughput nature of MS approaches and the sensitivity of molecular biology approaches like ELISA. A targeted mass spectrometry-based proteomics experiment focuses on detecting hypothesis driven proteins or peptides from discovery-based-shotgun proteomics experiments or through available literature1,2. Multiple Reaction Monitoring (MRM) is one such targeted MS approach that uses a tandem quadrupole mass spectrometer for accurate detection and quantification of proteins/peptides from complex samples. The technique offers higher sensitivity and specificity despite requiring the use of a low-resolution instrument.
A quadrupole is made of 4 parallel rods, with each rod connected to the diagonally opposite rod. A fluctuating field is created between the quadrupole rods by applying alternating RF and DC voltages. The trajectory of the ions inside the quadrupole is influenced by the presence of the same voltages across opposite rods. By applying the RF to DC voltage, the trajectory of the ions can be stabilized. It is this property of the quadrupole that allows it to be used as a mass filter which can selectively let specific ions to pass through. Depending on the need, a quadrupole can be operated in either the static mode or the scanning mode. The static mode allows only ions with a specified m/z to pass through, making the mode highly selective and specific to the ion of interest. The scanning mode on the other hand allows ions across the entire m/z range to pass through. Thus, tandem quadrupole mass spectrometers can operate in 4 possible ways: i) the first quadrupole operating in the static mode while the second operating in scanning mode; ii) the first quadrupole operating in the scanning mode while the second operating in the static mode; iii) both quadrupoles operating in the scanning mode; and iv) both quadrupoles operating in static mode3. In a typical MRM experiment, both the quadrupoles operate in the static mode allowing specific precursors and their resulting products after fragmentation to be monitored. This makes the technique very sensitive and selective allowing accurate quantification.
For molecular biologists, the human brain tissue and its cells are a treasure trove. These remarkable units of an ever-interesting organ of the human body can provide molecular and cellular insights into its functioning. Proteomic investigations of the brain tissue can not only help us understand the systemic functioning of a healthy brain but also the cellular pathways that get dysregulated when inflicted by some disease4. However, the brain tissue with all its heterogeneity is a very complex organ to analyze and requires a concerted approach for a better understanding of the changes at the molecular level. The following work describes the entire workflow starting right from extracting proteins from brain tissue, creating and optimising the methods for MRM assay, to validation of the targets (Figure 1). Here, we have systematically highlighted the major steps involved in a successful MRM based experiment using human brain tissue with an aim to introduce the technique and its challenges to a broader research community.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile (MS grade)</td><td>Fisher Scientific</td><td>A/0620/21</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>HiMedia</td><td>TC194-25G</td><td></td></tr><tr><td>Calcium chloride</td><td>Fischer Scienific</td><td>BP510-500</td><td></td></tr><tr><td>Formic acid (MS grade)</td><td>Fisher Scientific</td><td>147930250</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>1149-25G</td><td></td></tr><tr><td>Isopropanol (MS grade)</td><td>Fisher Scientific</td><td>Q13827</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Fischer Scienific</td><td>BP214-500</td><td></td></tr><tr><td>Methanol (MS grade)</td><td>Fisher Scientific</td><td>A456-4</td><td></td></tr><tr><td>MS grade water</td><td>Pierce</td><td>51140</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>HiMedia</td><td>TL1006-500ML</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche Diagnostics</td><td>11873580001</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>DF6D661300</td><td></td></tr><tr><td>TCEP</td><td>Sigma</td><td>646547</td><td></td></tr><tr><td>Tris Base</td><td>Merck</td><td>648310</td><td></td></tr><tr><td>Trypsin (MS grade)</td><td>Pierce</td><td>90058</td><td></td></tr><tr><td>Urea</td><td>Merck</td><td>MB1D691237</td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hypersil Gold C18 column</td><td>Thermo</td><td>25002-102130</td><td></td></tr><tr><td>Micropipettes</td><td>Gilson</td><td>F167380</td><td></td></tr><tr><td>Stage tips</td><td>MilliPore</td><td>ZTC18M008</td><td></td></tr><tr><td>Zirconia/Silica beads</td><td>BioSpec products</td><td>11079110z</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bead beater (Homogeniser)</td><td>Bertin Minilys</td><td>P000673-MLYS0-A</td><td></td></tr><tr><td>Microplate reader (spectrophotometer)</td><td>Thermo</td><td>MultiSkan Go</td><td></td></tr><tr><td>pH meter</td><td>Eutech</td><td>CyberScan pH 510</td><td></td></tr><tr><td>Probe Sonicator</td><td>Sonics Materials, Inc</td><td>VCX 130</td><td></td></tr><tr><td>Shaking Drybath</td><td>Thermo</td><td>88880028</td><td></td></tr><tr><td>TSQ Altis mass spectrometer</td><td>Thermo</td><td>TSQ02-10002</td><td></td></tr><tr><td>uHPLC - Vanquish</td><td>Thermo</td><td>VQF01-20001</td><td></td></tr><tr><td>Vacuum concentrator</td><td>Thermo</td><td>Savant ISS 110</td><td></td></tr></tbody></table>

quantitative proteomics workflow using multiple reaction monitoring based detection of proteins from human brain tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

We performed relative quantification of 3 proteins from 10 samples, 5 samples from each group of patients with abnormalities in the brain. These proteins included Apolipoprotein A-I (APOA-I), Vimentin (VIM) and Nicotinamide phosphoribosyltransferase (NAMPT) which are known to perform diverse roles in the brain cells. Post-run analysis of the data was performed using Skyline-daily (Ver 20.2.1.286). A total of 10 peptides corresponding to 3 proteins were monitored. These included 3 peptides for APOA-I, 4 peptides for VIM a...

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Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

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4.4K Views.  Indian Institute of Technology Bombay. Gerichte proteomische benaderingen worden snel de methode bij uitstek voor validatie van eiwitten uit op proteomics gebaseerde experimenten op korte termijn of uit op moleculaire biologie gebaseerde experimenten. Meervoudige reactiebewaking is zo'n gerichte aanpak die steeds vaker wordt gebruikt om eiwitten uit biologische monsters te detecteren en te kwantificeren. In deze studie hebben we de verschillende stappen doorlopen die betrokken zijn bij het detecteren en kwantificeren van eiwitten ...

Video: Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Chemistry

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Medicine

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

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