The authors thank all other lab and facility members for helpful discussion. This work was partially supported by NI H grants R01GM114260, R01AG047928, R01AG053987, and ALSAC. The MS analysis was performed in the St. Jude Children&#39;s Research Hospital Proteomics Facility, partially supported by NIH Cancer Center Support grant P30CA021765. The authors thank Nisha Badders for help with editing the manuscript.

CAUTION: Please consult all relevant safety data sheets (i.e., MSDS) before use. Please use all appropriate safety practices when performing this protocol.
NOTE: A TMT 10-plex isobaric label reagent set is used in this protocol for the proteome quantitation of 10 samples.
1. Preparation of Cells/Tissues
NOTE: It is critical to collect samples in minimal time at low temperature to keep proteins in their original biological state.</p...

<ol>
	<li>Pagala, V. 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=Quantitative+protein+analysis+by+mass+spectrometry.">Quantitative protein analysis by mass spectrometry.</a> Methods Mol Biol. 1278, 281-305 (2015).</li><li>Altelaar, A. F., Munoz, J., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+proteomics:+towards+an+integrative+view+of+proteome+dynamics.">Next-generation proteomics: towards an integrative view of proteome dynamics.</a> Nat Rev Genet. 14 (1), 35-48 (2013).</li><li>Rauniyar, N., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isobaric+labeling-based+relative+quantification+in+shotgun+proteomics.">Isobaric labeling-based relative quantification in shotgun proteomics.</a> J Proteome Res. 13 (12), 5293-5309 (2014).</li><li>Ross, P. L., 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=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Aebersold, R., 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=Mass-spectrometric+exploration+of+proteome+structure+and+function.">Mass-spectrometric exploration of proteome structure and function.</a> Nature. 537 (7620), 347-355 (2016).</li><li>Bai, 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=Deep+Profiling+of+Proteome+and+Phosphoproteome+by+Isobaric+Labeling.">Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling.</a> Extensive Liquid Chromatography, and Mass Spectrometry. Methods Enzymol. 585, 377-395 (2017).</li><li>McAlister, G. 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=Increasing+the+multiplexing+capacity+of+TMTs+using+reporter+ion+isotopologues+with+isobaric+masses.">Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.</a> Anal Chem. 84 (17), 7469-7478 (2012).</li><li>Everley, R. A., Kunz, R. C., McAllister, F. E., Gygi, S. 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We describe a high-throughput protocol for the quantitation of proteins with a 10-plex isobaric labeling strategy, which has been implemented successfully in several publications12,13,14,32. In this protocol, we can analyze up to 10 different biological protein samples in 1 experiment. We can routinely identify and quantitate well over 10,000 proteins with high confidence. Although isobaric lab...

Advances in next-generation sequencing technology have led to a new landscape for studying biological systems and human disease. This has permitted a large number of measurements of the genome, transcriptome, proteome, metabolome, and other molecular systems to become tangible. Mass spectrometry (MS) is one of the most sensitive methods in analytical chemistry, and its application in proteomics has rapidly expanded after the sequencing of the human genome. In the proteomics field, the past few years have yielded major technical advances in MS-based quantitative analyses, including isobaric labeling and multiplexing capability combined with extensive liquid chromatography, in addition to instrumentation advances, allowing for faster, more accurate measurements with less sample material required. Quantitative proteomics have become a mainstream approach for profiling tens of thousands of proteins and posttranslational modifications in highly complex biological samples1,2,3,4,5,6.
Multiplexed isobaric labeling methods such as isobaric tag for relative and absolute quantitation (i.e., iTRAQ) and tandem mass tag (TMT) MS have greatly improved sample throughput and increased the number of samples that can be analyzed in a single experiment1,6,7,8. Along with other MS-based quantitation methods, such as label-free quantitation and stable isotope labeling with amino acids in cell culture (i.e., SILAC), the potential of these techniques in the proteomics field is considerable9,10,11. For example, the TMT method permits 10 protein samples to be analyzed together in 1 experiment by using 10-plex reagents. These structurally identical TMT tags have the same overall mass, but heavy isotopes are differentially distributed on carbon or nitrogen atoms, resulting in a unique reporter ion during MS/MS fragmentation of each tag, thereby enabling relative quantitation between the 10 samples. The TMT strategy is routinely applied to study biological pathways, disease progression, and cellular processes12,13,14.
Substantial technical improvements have enhanced liquid chromatography (LC) &#8211;MS/MS systems, both in terms of LC separations and MS parameters, to maximize protein identification without sacrificing quantitation accuracy. First-dimension separation of peptides by a separation technique with high orthogonality to the second dimension is critical in this type of shotgun proteomics method to achieve maximum results20. High-pH reversed-phase liquid chromatography (RPLC) provides better performance than does conventional strong cation-exchange chromatography20. When high-pH RPLC is combined with a second dimension of low-pH RPLC, both analytical dynamic range and protein coverage are improved, resulting in the ability to identify the bulk of expressed proteins when performing whole-proteome analyses15,16,17,18. Other technical advances include small C18 particles (1.9 &#181;m) and extended long column (~1 m)19. Furthermore, other notable improvements include new versions of mass spectrometers with rapid scan rates, improved sensitivity and resolution20, and sophisticated bioinformatics pipelines for MS data mining21.
Here, we describe a detailed protocol that incorporates the most recent methodologies with modifications to improve both sensitivity and throughput, while focusing on quality control mechanisms throughout the experiment. The protocol includes protein extraction and digestion, TMT 10-plex labeling, basic pH and acid pH RPLC fractionation, high-resolution MS detection, and MS data processing (Figure 1). Moreover, we implement several quality control steps for troubleshooting and evaluating experimental variation. This detailed protocol is intended to help researchers new to the field routinely identify and accurately quantitate thousands of proteins from a lysate or tissue.

<table>
	<tbody>
		<tr>
			<td>1220 LC system</td>
			<td>Agilent</td>
			<td>G4288B</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Hydroxylamine</td>
			<td>Thermo Scientific</td>
			<td>90115</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH015-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender</td>
			<td>Next Advance</td>
			<td>BB24-AU</td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly Portfolio Heater</td>
			<td>Phoenix S&#38;T</td>
			<td>PST-BPH-20</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 tips</td>
			<td>Harvard Apparatus</td>
			<td>74-4607</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>41648</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector</td>
			<td>Gilson</td>
			<td>FC203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>Next Advance</td>
			<td>GB05</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lys-C</td>
			<td>Wako</td>
			<td>125-05061</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH230-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>Q Exactive HF</td>
			<td></td>
		</tr>
		<tr>
			<td>nanoflow UPLC</td>
			<td>Thermo Scientific</td>
			<td>Ultimate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur C18 resin, 1.9um</td>
			<td>Dr. Maisch GmbH</td>
			<td>r119.aq.0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Self Pck Columns</td>
			<td>New Objective</td>
			<td>PF360-75-15-N-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedva</td>
			<td>Thermo Scientific</td>
			<td>SPD11V</td>
			<td></td>
		</tr>
		<tr>
			<td>TMT 10plex Isobaric label reagent</td>
			<td>Thermo Scientific</td>
			<td>90110</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Applied Biosystems</td>
			<td>400003</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V511C</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Xbridge Column C18 column</td>
			<td>Waters</td>
			<td>186003943</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziptips C18</td>
			<td>Millipore</td>
			<td>ZTC18S096</td>
			<td></td>
		</tr>
		<tr>
			<td>SepPak 1cc 50mg</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 used a previously described cross-species peptide mix to systematically analyze the effect of ratio compression in 3 major protocol steps, including pre-MS fractionation, MS settings, and post-MS correction23. The pre-MS fractionation was evaluated and optimized by using a combination of basic pH RPLC and acidic pH RPLC. For post-MS analysis, only species-specific peptides were considered. We used this interference model to examine a number of parameters in LC/L...

Watch this Scientific Journal Video about Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification at JoVE.com

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

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) ...

The overall goal of this experiment is to identify and accurately quantitate over 10, 000 proteins from cell or tissue lysate across different drug treatments, time courses, or biological conditions. This method can help answer key questions in the field of medicine or biology such as what proteins are changing in response to a drug or other biological stimulus. The main advantage of this technique is that it has the ability to accurately quantitate over 70%of the expressed proteome over 10 different experimental conditions.The implication of this technology can be extended to the diagnosis of human diseases through potential biomarker discovery. Visual demonstration of this method is critical as optimal cell lysis and fluid estimation play a key role in downstream digestion, labeling, and separation procedures. To begin this protocol, obtain cultured cells of interest.Wash the cells twice using 10 milliliters of PBS for each wash. Scrape and collect the washed cells in a 1.5 milliliter tube containing one milliliter of PBS. Centrifuge at 600 times g and four degrees Celsius for five minutes.Remove the supernatant and store at minus 80 degrees Celsius until ready to lyse the cells. Collect and weigh the desired tissues quickly after dissection. After this, immediately store them in liquid nitrogen and store at minus 80 degrees Celsius.Prepare the lysis buffer on the day of the experiment as outlined in the text protocol. Add lysis buffer to the frozen sample such that the buffer to sample ratio is 10 to one and the final protein concentration is between five and 10 milligrams per milliliter. Next, add glass beads and use a blender to lyse the samples at four degrees Celsius and speed eight by blending for 30 seconds and then resting for five seconds, repeating this five times or until the samples are homogenized.Measure the protein concentration using a standard protein quantitation assay or a Coomassie-stained SDS polyacrylamide gel with BSA as a standard. After this, add 100%acetonitrile such that the final concentration is 10%and Lys-C protease is at an enzyme to substrate ratio of one to 100. Incubate at room temperature for two hours to allow protein digestion to occur.Then add DTT such that the final concentration is one millimolar. Incubate at room temperature for one hour. Next, add 50 millimolar HEPES until the urea concentration in the samples is diluted to two molar.Add trypsin to each sample at a trypsin to protein ratio of one to 50. Incubate at room temperature for at least three hours. Next, add DTT until its concentration is once again one millimolar and incubate at room temperature for two hours.Add iodoacetamide such that its final concentration is 10 millimolar. Incubate in the dark for 30 minutes at room temperature. After this, quench any unreacted iodoacetamide by adding DTT such that its final concentration is 30 millimolar.Incubate at room temperature for 30 minutes. Check the efficiency of the trypsin digestion as outlined in the text protocol. Next, add TFA to each sample such that the final concentration is 1%Using a pH strip, measure the pH of each sample to verify it is between two and three.Add additional TFA drop wise if needed to reach an unacceptable pH. Centrifuge the samples at 20, 000 times g and room temperature for 10 minutes. Collect the supernatant and load the samples onto prepared spin columns.Centrifuge at 100 times g for three minutes or until the sample has passed completely through the column to bind the samples. Next, add 0.5 milliliters of 0.1%TFA to each column. Centrifuge at 500 times g for 30 seconds.After this, add 125 microliters of a solution containing 60%acetonitrile and 0.1 TFA to each column. Centrifuge at 100 times g for three minutes to elute the peptides off the column. Reconstitute each desalted peptide sample in 50 microliters of 50 millimolar HEPES.Using a pH strip, verify that the pH of each sample is between seven and eight. Next, dissolve the TMT reagents in anhydrous acetonitrile, add them to the samples, and then incubate at room temperature for one hour. After this, use 10 microliter pipette tips embedded with chromatography media to desalt one microgram of each sample and analyze the labeling efficiency as outlined in the text protocol.Examine six to 10 separate peptides to ensure that unlabeled peptides are not detected in the labeled samples. Then, mix approximately two microliters of each sample together. Use 10 microliter pipette tips embedded with chromatography media to desalt this mixture.Analyze the samples by LC-MS/MS as outlined in the text protocol. To begin, solubilize the desalted pooled TMT-labeled peptide sample in 65 microliters of buffer A.Use a pH strip to verify that the pH is approximately 8.0. If the sample is still acidic, use ammonium hydroxide to adjust the pH as necessary.Next, fractionate the sample as outlined in the text protocol. Set the fraction collector to collect fractions every two minutes including loading time. Set the flow rate to 0.4 milliliters per minute.Collect a total of 80 fractions. Then, use a vacuum concentrator to completely dry every other sample. Using LC-MS/MS, analyze all 80 fractions to achieve ultra deep proteome coverage.First, pack 1.9 micrometer C18 resin into 75 micrometer inner diameter empty columns reaching a bed volume of approximately 1.3 microliters. Coil the column two to three times inside a butterfly portfolio heater to ensure the entire length is heated. Tape the column to the inside of the heater making sure not to tape over the heater's temperature sensor.Then, heat the column to 65 degrees Celsius. Run 100 nanograms of rat brain peptides through the LC-MS/MS system to assess the system's quality. Repeat this assessment one additional time.After this, load approximately 0.2 micrograms of reconstituted peptides on the column while flowing 5%buffer A.Elute the peptides off the column and analyze with the mass spectrometer as outlined in the text protocol. In this study, a high throughput method for the quantitation of proteins with a 10-plex isobaric labeling strategy is described. TMT labeling efficiency is examined using LC-MS/MS to analyze TMT-labeled samples and corresponding unlabeled samples.As can be seen, the unlabeled peptide peak is found only in the unlabeled sample while the TMT-labeled peptide peak is found only in the labeled sample. Next, the effect of the MS2 isolation window on interference is evaluated. All MS2 scans are designated either as a clean or a noisy scan.While the clean scans exhibit y1 ions of only lysine, the noisy scans exhibit them in both lysine and arginine. Representative interference removal using E.coli peptides individually labeled with three different TMT reagents is shown here. The summed relative intensities reveal that y1 ion-based correction is more accurate for TMT-based quantitation.Once mastered, this protocol can be completed in four to five weeks if done properly. While attempting this procedure, it is very important to make sure all the quality control steps are done completely to ensure the final dataset is as accurate as possible. Adapting this procedure to focus on post-translational modifications can answer other questions such as how ubiquitination, phosphorylation, or acetylation is changing in response to disease progression or drug treatment.After its development, the technology paved the way for researchers in the product field to explore the protein changes in cells and tissues. After watching this video, you should have a very good understanding of how to identify and accurately quantitate over 10, 000 proteins from a mammalian cell or tissue. Don't forget that working with high-pressure UPLC and few silica columns can be extremely hazardous and precautions such as wearing safety goggles should always be taken while performing this procedure.We first had the idea of this method when we began to address the known problem of ratio compression which occurs in isobaric labeling. We have realized extensive fractionation is a great way to not only alleviate this problem, but also increases the identification of proteins and peptides which can further increase and adapts of a proteome analysis.

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Research

JoVE Journal

Biochemistry

11.8K vistas.  St. Jude Children's Research Hospital. The overall goal of this experiment is to identify and accurately quantitate over 10, 000 proteins from cell or tissue lysate across different drug treatments, time courses, or biological conditions. This method can help answer key questions in the field of medicine or biology such as what proteins are changing in response to a drug or other biological stimulus. The main advantage of this technique is that it has the ability to accurately quantitate over 70%of the expressed proteome over 10 d...