Name: quantitative proteomics workflow using multiple reaction monitoring based detection of proteins from human brain tissue
Uploaded: 2021-08-28T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue at JoVE.com

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 tis...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+applications+of+liquid+chromatography-mass+spectrometry+in+clinical+biochemistry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primer+on+Concepts+and+Applications+of+Proteomics+in+Neuroscience.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+by+spectrophotometry+at+205+nm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+SRMAtlas:+A+Resource+of+Targeted+Assays+to+Quantify+the+Complete+Human+Proteome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+of+proteins+and+phosphoproteins+from+cell+lysates+by+tandem+MS.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+iRT,+a+normalized+retention+time+for+more+targeted+measurement+of+peptides.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+peptides+and+proteins+in+biomedicine+by+targeted+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+targeted+proteomics-based+pipeline+for+verification+of+biomarkers+in+plasma.">A targeted proteomics-based pipeline for verification of biomarkers in plasma.</a> Nature Biotechnology. , (2011).</li><li>H&#252;ttenhain, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+quantification+of+cancer-associated+proteins+in+body+fluids+using+targeted+proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRM/MRM+targeted+proteomics+as+a+tool+for+biomarker+validation+and+absolute+quantification+in+human+urine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+directly+interacts+with+amyloid+precursor+protein+and+inhibits+A&#946;+aggregation+and+toxicity.">Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits A&#946; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin+participates+in+microglia+activation+and+neurotoxicity+in+cerebral+ischemia.">Vimentin participates in microglia activation and neurotoxicity in cerebral ischemia.</a> Journal of Neurochemistry. , (2012).</li><li>Liu, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+Phosphoribosyltransferase+May+Be+Involved+in+Age-Related+Brain+Diseases.">Nicotinamide Phosphoribosyltransferase May Be Involved in Age-Related Brain Diseases.</a> PLoS ONE. , (2012).</li><li>Abbatiello, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+guidelines+for+publication+of+manuscripts+describing+development+and+application+of+targeted+mass+spectrometry+measurements+of+peptides+and+proteins.">New guidelines for publication of manuscripts describing development and application of targeted mass spectrometry measurements of peptides and proteins.</a> Molecular and Cellular Proteomics. 16 (3), 327-328 (2017).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</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>Supplies</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>Equipment</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...

Watch this Scientific Journal Video about Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue at JoVE.com

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 ...

Targeted proteomic approaches are fast becoming the method of choice for validation of proteins from short-term proteomics-based experiments or from molecular biology-based experiments. Multiple reaction monitoring is one such targeted approach which is increasingly being used to detect and quantify proteins from biological samples. In this study we have walked through the various steps involved in detecting and quantifying proteins from human brain tissue with an intention of introducing the readers to the basic requirements of an MRM experiment.Weigh around 50 mg of brain tissue, and wash the tissue with 300 microliters of 1x phosphate-buffered saline. This step is performed to remove any blood on the external surface of the tissue, and hence it is advisable to remove as much blood as possible. Following washes with PBS, add 300 microliters of lysis buffer to the tube containing the tissue.Lyse the tissue using a probe sonicator while keeping the tube on an ice bath. Continue with tissue homogenization using a bead beater to completely lyse the tissue. Centrifuge the contents of the tube at 6000 g for 15 minutes at four degrees centigrade.Transfer the supernatant into a fresh tube and mix homogeneously. Take a small aliquot of the supernatant, and quantify the protein content using a standard protein assay. Here we show an example of protein estimation using Bradford Assay.Take 50 micrograms of protein in a microcentrifuge tube and reduce the contents by adding TCEP such that the final concentration is 20 millimolar. Incubate the contents at 37 degrees for one hour. Following incubation, alkylate the reduced proteins by adding iodoacetamide to the tube, such that the final concentration is 40 millimolar.Incubate the tube in dark for 30 minutes. Now add Buffer B to the tube contained the reduced and alkylated proteins such that the final concentration of urea is less than one molar. Add trypsin in 1 as to 30 enzyme-to-protein ratio and incubate the tubes overnight at 37 degrees with constant shaking.Following digestion, concentrate the digested peptides in a vacuum concentrator. At this step, the peptides can either be reconstituted and desalted or stored at minus 80 for future use. Desalting, or peptide clean-up, is essential before loading the samples on any LC-MS MS.Salts and other contaminants in the sample can clog the column and affect the sensitivity of the instrument in the long run.This process can be performed using commercially available C18 STAGE tips or columns. Here we have depicted the workflow for peptide desalting using a C18 STAGE tip. Following desalting, dry the peptides in a vacuum concentrator.Reconstitute these clean peptides and proceed for quantification using the Scopes method. For this, load two microliters of the sample in a microdrop plate and calculate the concentration as shown in the slide. Transition list preparation is a crucial step in an SRM or MRM experiment.A transition list contains all the information required by the mass spectrometer to monitor the transition. This include the m by z values of the precursor and product ions, collision energy of the transition, polarity of the instrument, and the time range for data acquisition. Prepare the transition list using Skyline.Prepare a background proteome using Human Proteome Fasta file from UniProt and ensure it is selected under the Background tab of Peptide Settings. In the Filter tab, set a minimum length of 8 and maximum length of 25. Continue to use the default settings if there are no other modifications in the peptides.Once the peptide settings have been made, click on Transition Settings. Under the Filter tab, set Precursor charges as 2 and 3. Set Ion charges as 1 and set Ion types to y.Product ions can be selected depending on the user's choice. Leave all the parameters as default. Now insert the accession IDs of the selected target proteins.This can be done by clicking on Edit tab then selecting Insert and finally pasting the accession IDs. The IDs will be mapped to the proteins in the background proteome and a transition list created based on the peptide and transition settings set earlier. Export this transition list.Ensure that the right instrument is selected by clicking on the Instrument type dropdown option. In this case, the instrument is Thermo Altis. Since the undefined transition list has too many transitions, keep a limit of only 350 transitions per list and export as multiple methods.A binary solvent system is used. Solvent A is 1%FA and Solvent B is 80%ACN. Keep the flow rate at 450 microliter per minute and the gradient as shown.Set column compartment temperature at 45 degrees Celsius. For further details about column and LC system, refer text. Ensure the MS settings in your TSQ Altis are as shown here.Import a single transition list to create a new method. We suggest to keep the resolution for quadrupole 1 and quadrupole 3 at 7. These can be further optimized if required.To ensure consistency in runs and consequently the data quality, prepare your run sequence as shown. Start with a couple of blanks, refer to the text for composition of blank mixture, followed by a QC standard light BSA of known concentration to monitor any variation in the day-wise response of the instrument. The QC done should be just before the samples.Next line up all the samples with blanks in between. Inject equal volumes of each sample, accounting for 25 to 1 nanogram of peptides per injection. To make a refined method, first run the undefined methods against whole samples.Import results into Skyline and manually check each peptide. For each peptide, select one precursor showing consistently good peaks in all the samples. Delete precursors and peptides that are not showing good peaks.Make sure all the peaks are annotated properly. To select the right peak and further refine the transitions under each precursor, a library can be used. A library is a set of MS-MS spectral data matched with peptides and transitions of the current experimental data.Pick peaks that have DART P values closer to 1 and remove transitions that do not match the library. A DART P value denotes how good the match is between the experimental peak and library peak. After one or two rounds of optimization, a refined list will be ready.This refined list can be run for individual samples. Import the refined runs into Skyline and annotate peaks properly. Use the Annotation tab under Document Settings to annotate each sample in the document grid carefully.Use the Group Comparison feature to create comparisons between Condition 1 and Condition 2 of your experiment. This will give table with adjusted P values and fold change values between the two groups. To our knowledge, this is the first study employing the use of MRM to detect peptides for the proteins Apolipoprotein A-1, Vimentin, and Nicotinamide phosphoribosyltransferase in human brain tissue samples.Optimizing various parameters such as LC gradient, dwell time, cycle time, and collision energy can greatly influence the success of an MRM experiment. We believe that this technique can be used to develop patterns of biomarkers with ability to distinguish between different clinical conditions and diseases.

Research

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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

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