The authors acknowledge the University of Pittsburgh Start-up Funds and NIH, NIGMS R01 grant (GM 117191-01) to RASR.

Ethics Statement: Mice were purchased from an independent, non-profit biomedical research institution and housed in the Division of Laboratory Animal Resources at the University of Pittsburgh. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.
1. Protein Extraction and Generation of Peptides for Chemical-tagging
<ol>
	<li>Extract protein from tissue, cells, or bodily fluids.
	<ol>
		<li>Homogenize 60-90 mg of tissue (e.g. brain, heart, and liver) in phosphate buffer saline (1x PBS) with 8 M urea (500 &#181;L) using a mechanical homogenizer. Protease or phosphatase inhibitors may be added to the buffer if necessary. In this project, they were not used. Use the following parameters for the homogenizer: lysing matrix A beads, 4 m/s, 20 s. Heart tissue may require up to 9 cycles of homogenization. 
		NOTE: Protease or phosphatase inhibitors may be added to the buffer if necessary. They were not used in this study.
		<ol>
			<li>Remove tissue homogenate from the lysing tube and transfer to a micro-centrifuge tube. Rinse lysing tubes with 100-500 &#181;L of PBS with 8 M urea and combine the rinse solution with tissue homogenate.</li>
		</ol>
		</li>
		<li>Centrifuge the homogenized tissues (9,447 x g, 4 &#176;C, 15 min) and collect the supernatant.</li>
	</ol>
	</li>
	<li>Determine the protein concentration using a bicinchoninic acid (BCA) assay according to manufacturer&#39;s instructions. 
	NOTE: Further dilution with buffer may be necessary if the protein concentration is too high. The resulting concentrations for samples from these tissues were in between 6-13 &#181;g/&#181;L.
	<ol>
		<li>Optional: Add an internal quality control standard (e.g. bovine alpha casein or other exogenous protein) with the ratio 1 &#181;g standard:100 &#181;g protein sample.</li>
	</ol>
	</li>
</ol>
2. Sample Digestion
<ol>
	<li>Add 100 &#181;g (100 x 10-6 g) of protein from each sample to individually labeled micro-centrifuge tubes. The volume is dependent on the protein concentration (see step 1.2).</li>
	<li>Add dithiothreitol (DTT) (40:1 reagent to sample mol ratio) to each sample. Incubate at 37 &#176;C for 2 h. The calculation for the molar ratio is based off the protein mass of bovine serum albumin (BSA), which is 66.5 x 103 g/mol.</li>
	<li>Add iodoacetamide (IAM) (80:1 reagent to sample mol ratio) to each sample. Incubate on ice in the dark for 2 h. This reaction is performed on ice for 2 h to prevent side reactions.</li>
	<li>Add L-cysteine (40:1 reagent to sample mol ratio) to each sample. Incubate at room temperature for 30 min.</li>
	<li>Add 20 mM Tris buffer with 10 mM CaCl2 (pH 8.2) to dilute urea to a final concentration of 2 M.</li>
	<li>Add L-1-tosylamido-2 phenylethyl chloromethyl ketone (TPCK)-treated trypsin (50:1 substrate to enzyme mol ratio) to each sample and incubate at 37 &#176;C for 24 h.</li>
	<li>Quench the protein digestion by flash-freezing the sample in liquid nitrogen and store at -80 &#176;C until further processing.</li>
</ol>
3. Sample Desalting
<ol>
	<li>Re-acidify the samples by adding formic acid (FA). Add enough FA to obtain a final concentration of 0.1%. If samples are originally at -80 &#176;C, thaw samples on ice before re-acidification.</li>
	<li>De-salt the peptides using C18 hydrophilic lipophilic balanced (HLB) 10 mg cartridges as previously described16.</li>
	<li>Dry the samples by vacuum centrifugation (5 Torr, 45 &#176;C, 77 x g) to a volume of ~10 &#181;L.</li>
</ol>
4. Dimethylation Labeling (N-termini)
<ol>
	<li>Reconstitute peptides in 1% acetic acid (0.25 &#181;g/&#181;L) dissolved in high performance liquid chromatography (HPLC) or nano-pure grade water. Remove a 50 &#181;g portion to a new micro-centrifuge tube (1.5 mL) and store the remainder at -80 &#176;C.</li>
	<li>Add 8 &#181;L of 60 mM (4%) CH2O (37% wt/v) or 60 mM (4%) 13CD2O (20% wt/v) to the samples for labeling with light or heavy dimethylation, respectively. Typically, 4 &#181;L of reagent is added per 25 &#181;g of peptides (Steps 4.2, 4.3, 4.6).</li>
	<li>Add 8 &#181;L of 24 mM NaBH3CN or 24 mM NaBD3CN to the samples labeled with light or heavy dimethylation, respectively.</li>
	<li>Vortex and shake on a tube shaker for ~10 min at room temperature.</li>
	<li>Quench the reactions by adding 16 &#181;L of 1% ammonia (~28-30% v/v) for 5 min. Typically, 8 &#181;L of reagent is added per 25 &#181;g of peptides.</li>
	<li>Re-acidify the reaction mixtures by adding 8 &#181;L of 5% FA (98% v/v).</li>
	<li>Combine the light and heavy dimethylated peptides (Figure 1) to generate a total of six samples. See Table 1 for a description of sample tagging and pooling used for this study.</li>
	<li>Perform sample desalting according to steps 3.2 and 3.3.</li>
</ol>
5. Isobaric Tagging (Lys residues)
<ol>
	<li>Reconstitute 100 &#181;g of dimethylated peptides (1 &#181;g/&#181;L) in 100 mM tri-ethyl ammonium bicarbonate (TEAB) buffer (pH ~8.5).</li>
	<li>Prepare isobaric reagents as stated in the manufacturer&#39;s protocol (see Table of Materials/Reagents).</li>
	<li>Add solubilized isobaric reagents (41 &#181;L) to peptides and vortex for ~10 s. Shake the samples on a micro-centrifuge tube shaker for ~1 h. Quench the reactions with 8 &#181;L hydroxylamine (10% w/v) and incubate the samples for 15 min at room temperature.</li>
	<li>Pool the six labeled samples into a single mixture and desalt (Steps 3.1-3.3) or store at -80 &#176;C until further use. 
	NOTE: For improving proteome coverage and depth, a multi-dimensional separation strategy is suggested such as strong cation exchange (SCX).</li>
</ol>
6. Strong Cation Exchange
<ol>
	<li>Perform SCX as per the manufacturer&#39;s protocol (see Materials Table).
	<ol>
		<li>Prepare ~1 mL of ammonium formate solutions (20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, 250 mM, &#38; 500 mM) with 10% acetonitrile (ACN), 0.1% FA (pH 3).</li>
		<li>Remove the red cap from the top of the pipette tip and tap the pipette tip with packing slurry to ensure that the packing material is towards the bottom of the pipette tip. 
		NOTE: Critical: Do not throw away the pipette tip until the end of the protocol.</li>
		<li>Place the centrifuge adapter onto the top of a micro-centrifuge tube (2 mL) and secure the pipette tip inside.</li>
		<li>Add 150 &#181;L of SCX reconstitution buffer to pipette tips.</li>
		<li>Centrifuge the pipette tips for 6 min at room temperature (4,000 x g). Repeat this step two more times and discard the waste.</li>
		<li>Dissolve the peptides in 150 &#181;L of SCX reconstitution buffer. Ensure that the pH is 3.</li>
		<li>Add peptides to the pipette tips, centrifuge (see 6.1.5), and keep the eluent.</li>
		<li>Transfer the filter and pipette to a new micro-centrifuge tube (2 mL) and add 150 &#181;L of 20 mM ammonium formate solution. Centrifuge for 6 min at room temperature (4,000 x g) and keep the eluent.</li>
		<li>Repeat step 6.1.8 an additional seven times, using successive concentrations (i.e. 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, 250 mM and 500 mM) of ammonium formate.</li>
	</ol>
	</li>
	<li>Transfer the eight SCX fractions to micro-centrifuge tubes (1.5 mL) and concentrate them by using centrifugal evaporation (see step 3.3).</li>
</ol>
7. Liquid Chromatography&#8211;Tandem Mass Spectrometry (LC-MS/MS) and&#160;MS3
<ol>
	<li>Reconstitute the peptides in mass spectrometry grade water with 0.1% FA, filter, and place into an auto-sampler vial. Filter peptides as follows:
	<ol>
		<li>Add reconstituted peptides to a micro-centrifuge tube containing a 0.65 &#181;m filter (see Materials Table).</li>
		<li>Centrifuge peptides at 12,000 x g for 1 min. Remove filter, discard, and add filtered peptides into an auto-sampler vial.</li>
	</ol>
	</li>
	<li>Prepare the mobile phase buffers as follows: 3% (v/v) ACN with 0.1% FA (A) and 100% ACN with 0.1% FA (B).</li>
	<li>Inject 6 &#181;L of each SCX fraction sample onto a trap column packed to 2 cm with C18 material (5 &#181;m, 200 &#197; pore size). 
	NOTE: Sample cleaning on the trap is as follows: 3 min, 0% B; 3 &#181;L/min using a 2D liquid chromatography system (see Materials Table).</li>
	<li>Run the analytical separation method. Use a 75 &#181;m i.d. x 13.2 cm laser pulled-tip fused silica capillary column packed with C18 material (5 &#181;m, 100 &#197;). The gradient is: 0-5 min, 10% B; 5-40 min, 10-15% B; 40-90 min, 15-25% B; 90-115 min, 25-30% B; 115-130 min, 30-60% B; 130-135 min, 60-80% B; 135-145 min, 80% B, 145-150 min, 80-10% B; 150-180 min, 10% B; 300 nL/min, 180 min.</li>
	<li>Run the data acquisition for the mass spectrometer while the analytical separation method is running. 
	NOTE: The parameters for the MS survey scan are as follows: 400-1,600 m/z, 60,000 resolution, automatic gain control (AGC) target 1 x 106 ions, maximum injection time 500 ms. Parameters for CID&#8211;MS/MS are as follows: Top 1-7 or Top 8-14 data dependent acquisition (DDA), 3 m/z isolation width, 500 minimum signal, 35% normalized collision energy (NCE), 0.25 activation q, 10 ms activation time, 3 x 104 AGC, 50 ms maximum injection time. Parameters for HCD&#8211;MS3 are as follows: 200 minimum signal, 4 m/z isolation width, 60% NCE, 0.1 activation time, 3 x 105 AGC, 250 ms IT, 300-1,300 m/z MS/MS selection range, exclude un-fragmented parent ion.</li>
	<li>Perform each analysis in duplicate for both the top 1-7 and top 8-14 runs. 
	NOTE: For samples run on a newer model of an instrument containing an orbitrap or a tribrid mass spectrometer, DDA parameters will vary and can be optimized to include more MS/MS and MS3 scans. Additionally, for tribrid instruments, synchronous precursor selection (SPS)-MS3 should be employed.</li>
</ol>
8. Data Analysis16
<ol>
	<li>Search the RAW files using protein analysis software against an appropriate database, such as mouse Uniprot. 
	NOTE: It is important to create two workflows for each RAW file in order to search for light and heavy dimethylated peptides.</li>
	<li>Search the RAW files using the following parameters: trypsin with two missed cleavages, peptide mass range 300-6,000 Da, 15 ppm parent mass tolerance, 1 Da fragmentation tolerance. Static modifications: light dimethylation (+28.031, peptide N-terminus) or heavy dimethylation (+36.076 Da, peptide N-terminus), carbamidomethyl (+57.021 Da, C). 
	NOTE: Sometimes the heavy dimethylated peak is ~7 Da (+35.069 Da, peptide N-terminus) is from the light dimethylated peak and thus should be incorporated into the search workflow for heavy dimethylated peptides. Dynamic modifications: oxidation (+15.995 Da, M), 6-plex isobaric tag (229.163 Da, K). Include a decoy database search to yield false discovery rates (e.g. 1 and 5%) and a quantitation node to search for the reporter ion intensities of isobaric tags.</li>
	<li>Statistics
	<ol>
		<li>Export data into an electronic spreadsheet program in order to normalize protein reporter ion values. For each protein, divide either the median reporter ion ratios or raw reporter ion intensities by the median ratio of the internal standard or raw reporter ion intensities of the internal standard, respectively. Use the appropriate statistical software such as an electronic spreadsheet program, Perseus, or R to determine significant changes among sample conditions.</li>
	</ol>
	</li>
</ol>

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The authors have no competing interests.

cPILOT allows for the simultaneous measurement of more than 12 unique samples. In order to ensure successful tagging at both the N-terminus and lysine residues of peptides, it is imperative to have the correct pH for each set of reactions and to perform the dimethylation reaction first for peptide labeling. Selective dimethylation at the N-terminus is performed by having a pH at ~2.5 (&#177;0.2). This is achieved by exploiting the differences of the pKA&#39;s of the amino groups on lysine and the N-terminus. At pH 2.5, l...

Proteomics often involves the analysis of many samples used to better understand disease processes, enzyme kinetics, post-translational modifications, response to environmental stimuli, response to therapeutic treatments, biomarker discovery, or drug mechanisms. Quantitative methods can be employed to measure relative differences in protein levels across the samples and can be label-free or involve isotopic labeling (metabolic, chemical, or enzymatic). Stable isotope labeling methods have grown in popularity because they allow many samples to be analyzed simultaneously and are suitable for samples from different cells, tissues, bodily fluids, or whole organisms. Isotope labeling methods1,2,3,4,5,6,7 increase experimental throughput, while reducing acquisition time, costs, and experimental error. These methods use precursor mass spectra to measure relative abundances of proteins from peptide peaks. In contrast, isobaric tagging reagents8,9,10 generate reporter ions that are either detected in MS/MS or MS3 11 spectra and these peaks are used to report on relative abundances of proteins.
The current state-of-the-art in proteomics multiplexing is either a 10-plex12 or 12-plex isobaric tag analysis13. Enhanced sample multiplexing (i.e. &#62;10 samples) methods have been developed by our laboratory for tissues14,15,16,17, and by others for the analysis of cells18,19,20, tissues 21, or targeted peptides22. We developed an enhanced multiplexing technique called combined precursor isotopic labeling with isobaric tagging (cPILOT). Global cPILOT is useful for getting information about the relative concentrations of all proteins across different sample conditions (&#8805;12)14. Figure 1 shows a general cPILOT workflow. Tryptic or Lys-C peptides are selectively labeled at the N-terminus with dimethylation using low pH2 and at lysine residues with 6-plex reagents using high pH. This strategy doubles the number of samples that can be analyzed with isobaric reagents which helps to reduce experimental costs and additionally, reduces experimental steps and time.
cPILOT is flexible as we have developed other methods to study oxidative post-translational modifications, including 3-nitrotyrosine-modified proteins14 and cysteine containing peptides with S-nitrosylation (oxcyscPILOT)23. We have also developed an amino acid selective approach, cysteine cPILOT (cyscPILOT)17. MS3 acquisition with a top-ion11 or selective-y1-ion method15 can help reduce reporter ion interference and improve quantitative accuracy of cPILOT. The use of MS3 in the acquisition method requires a high-resolution instrument with an orbitrap mass analyzer although low resolution ion trap instruments may also work24.
Previously, cPILOT has been used to study liver proteins16 from an Alzheimer&#39;s disease mouse model. Here, we describe how to perform global cPILOT analysis using brain, heart, and liver homogenates to study the role of the periphery in Alzheimer&#39;s disease. This experiment incorporates biological replication. Because of the versatility of cPILOT, interested users can use the technique to study other tissues for a range of biological problems and systems.

<table><tbody><tr><td>Water - MS Grade</td><td>Fisher Scientific</td><td>W6-4</td><td>4 L quantity is not necessary</td></tr><tr><td>Acetonitrile - MS Grade</td><td>Fisher Scientific</td><td>A955-4</td><td>4 L quantity is not necessary</td></tr><tr><td>Acetic Acid</td><td>J.T. Baker</td><td>9508-01</td><td></td></tr><tr><td>Ammonium hydroxide solution (28 - 30%)</td><td>Sigma Aldrich</td><td>320145-500ML</td><td></td></tr><tr><td>Ammonium formate</td><td>Acros Organics</td><td>208-753-9</td><td></td></tr><tr><td>Formic Acid</td><td>Fluka Analytical</td><td>94318-250ML-F</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Pierce Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>Urea</td><td>Biorad</td><td>161-0731</td><td></td></tr><tr><td>Tris</td><td>Biorad</td><td>161-0716</td><td></td></tr><tr><td>Dithiothreiotol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Iodoacetamide (IAM)</td><td>Acros Organics</td><td>144-48-9</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Aldrich, Chemistry</td><td>168149-25G</td><td></td></tr><tr><td>L-1-tosylamido-2 phenylethyl cholormethyl ketone (TPCK)-treated Trypsin from bovine pancreas</td><td>Sigma Aldrich, Life Science</td><td>T1426-100MG</td><td></td></tr><tr><td>Formaldehyde (CH&lt;sub&gt;2&lt;/sub&gt;O) solution; 36.5 - 38% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich, Life Science</td><td>F8775-25ML</td><td></td></tr><tr><td>Formaldehyde (&lt;sup&gt;13&lt;/sup&gt;CD&lt;sub&gt;2&lt;/sub&gt;O) solution; 20 wt % in D&lt;sub&gt;2&lt;/sub&gt;O, 98 atom % D, 99 atom % &lt;sup&gt;13&lt;/sup&gt;C</td><td>Sigma Aldrich, Chemistry</td><td>596388-1G</td><td></td></tr><tr><td>Sodium Cyanoborohydride; reagent grade, 95%</td><td>Sigma Aldrich</td><td>156159-10G</td><td></td></tr><tr><td>Sodium Cyanoborodeuteride; 96 atom % D, 98% CP</td><td>Sigma Aldrich, Chemistry</td><td>190020-1G</td><td></td></tr><tr><td>Strong Cation Exchange (SCX) spin tips sample prep kit</td><td>Protea BioSciences</td><td>SP-155-24kit</td><td></td></tr><tr><td>Triethyl ammonium bicarbonate (TEAB) buffer</td><td>Sigma Aldrich, Life Science</td><td>T7408-100ML</td><td></td></tr><tr><td>Isobaric Tagging Kit (TMT 6 plex) - 6 reactions (1 x 0.8 mg)</td><td>Thermo Fisher Scientific</td><td>90061</td><td></td></tr><tr><td>Hydroxylamine hydrochloride</td><td>Sigma Aldrich, Chemistry</td><td>255580-100G</td><td></td></tr><tr><td>Standard vortex mixer</td><td>Fisher Scientific</td><td>2215365</td><td>any mixer can be used</td></tr><tr><td>Oasis HLB 1 cc (10 mg) extraction cartridges</td><td>Waters</td><td>186000383</td><td>These are C&lt;sub&gt;18&lt;/sub&gt; cartridges</td></tr><tr><td>Visiprep SPE vacuum manifold, DL (disposable liner), 24 port model</td><td>Sigma Aldrich</td><td>57265</td><td>A 12 port model is also sufficient</td></tr><tr><td>Speed-vac</td><td>Thermo Scientific</td><td>SPD1010</td><td>any brand of speed vac is sufficient</td></tr><tr><td>Water bath chamber</td><td>Thermo Scientific</td><td>2825/2826</td><td>Any brand of&amp;nbsp; a water bath chamber with controlled temperatures is sufficient.</td></tr><tr><td>Mechanical Homogenizer (&lt;em&gt;i.e.&lt;/em&gt; FastPrep-24 5G)</td><td>MP Biomedicals</td><td>116005500</td><td></td></tr><tr><td>Eksigent Nano LC - Ultra 2D with Nano LC AS-2 autosampler</td><td>Sciex</td><td>-</td><td>This model is no longer available. Any nano LC with an autosampler is sufficient.</td></tr><tr><td>LTQ Orbitrap Velos Mass Spectrometer</td><td>Thermo Scientific</td><td>-</td><td>This model is no longer available. Other high resolution instruments (&lt;em&gt;e.g.&lt;/em&gt; Orbitrap Elite, Orbitrap Fusion, or Orbitrap Fusion Lumos) can be used.</td></tr><tr><td>Protein software (&lt;em&gt;e.g.&lt;/em&gt; Proteome Discoverer)</td><td>Thermo Scientific</td><td>IQLAAEGABSFAKJMAUH&amp;nbsp;</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>AL54</td><td></td></tr><tr><td>Stir plate</td><td>VWR</td><td>12365-382</td><td>Any brand of stir plates are sufficient</td></tr><tr><td>pH meter (Tris compatiable)</td><td>Fisher Scientific (Accumet)</td><td>13-620-183</td><td>Any brand of a pH meter is sufficient</td></tr><tr><td>pH 10 buffer</td><td>Fisher Scientific</td><td>06-664-261</td><td>Any brand of pH buffer 10 is sufficient</td></tr><tr><td>pH 7 buffer</td><td>Fisher Scientific</td><td>06-664-260</td><td>Any brand pH buffer 7 is sufficient</td></tr><tr><td>1.5 mL eppendorf tubes, 500 pk</td><td>Fisher Scientific</td><td>05-408-129</td><td>Any brand of 1.5 mL eppendorf tubes are sufficient</td></tr><tr><td>0.6 mL eppendorf tubes, 500 pk</td><td>Fisher Scientific</td><td>04-408-120</td><td>Any brand of 0.6 mL eppendorf tubes are sufficient</td></tr><tr><td>0.65 &amp;micro;m Ultrafree MC DV centrifugal filter units</td><td>EMD Millipore</td><td>UFC30DV00</td><td></td></tr><tr><td>2 mL microcentrifuge tubes, 72 units</td><td>Thermo Scientific</td><td>69720</td><td></td></tr><tr><td>C&lt;sub&gt;18&lt;/sub&gt; packing material (5&amp;nbsp;&amp;micro;m, 100 &amp;Aring;)</td><td>Bruker</td><td>PM5/61100/000</td><td>This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient</td></tr><tr><td>C&lt;sub&gt;18&lt;/sub&gt; packing material (5&amp;nbsp;&amp;micro;m, 200 &amp;Aring;)</td><td>Bruker</td><td>PM5/61200/000</td><td>This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient</td></tr></tbody></table>

enhanced sample multiplexing of tissues using combined precursor isotopic labeling and isobaric tagging (cpilot)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

cPILOT uses amine-based chemistry to chemically label peptides at the N-terminus and lysine residues and enhances sample multiplexing capabilities. Figure 2 shows representative MS data that is obtained from a 12-plex cPILOT analysis of brain, heart, and liver tissues from an Alzheimer&#39;s disease mouse model and wild-type controls. As shown in Table 1, two biological replicates for the Alzheimer&#39;s disease and wild-type mice are included in this 12-...

Watch this Scientific Journal Video about Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT at JoVE.com

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

enhanced-sample-multiplexing-tissues-using-combined-precursor

Research

JoVE Journal

Biochemistry

6.9K Views.  University of Pittsburgh.  Gecombineerd voorloper isotopische labeling en isobare tagging (cPILOT) is een kwantitatieve proteomics strategie die steekproef multiplexing mogelijkheden van isobarisch labels verbetert. Dit protocol beschrijft de toepassing van cPILOT op weefsels van de ziekte van Alzheimer muismodel en wildtype controles. 

Video: Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

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) ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Cancer Research

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Chemistry

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

Bioengineering

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunology and Infection

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Biology

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Samenvatting

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