This work was partially supported by the National Institutes of Health (R01GM114260, R01AG047928, R01AG053987, RF1AG064909, and U54NS110435) and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in St. Jude Children&#8217;s Research Hospital&#8217;s Center of Proteomics and Metabolomics, which is partially supported by NIH Cancer Center Support Grant (P30CA021765). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Human tissues for the study were obtained with approvals from the Brain and Body Donation Program at Banner Sun Health Research Institute.
1. Protein extraction from tissue and quality control
NOTE: To reduce the impact of sample harvesting on the proteome, it is crucial to collect samples in minimal time at low temperature if possible31. This is especially important when analyzing posttranslational modifications as they typically are labile, f...

An optimized protocol for TMT16-based deep proteome profiling has been implemented successfully in earlier publications12,13,41. With this current protocol, more than 10,000 unique proteins from up to 16 different samples can be routinely quantified in a single experiment with high precision.
To obtain high-quality results, it is important to pay attention to critical steps throughout the protocol. In...

Rapid developments in mass spectrometry technology have enabled to achieve high sensitivity and deep proteome coverage in proteomics applications1,2. Despite these developments, sample multiplexing remains the bottleneck for researchers handling the analysis of a large sample cohort.
Multiplexed isobaric labeling techniques are extensively used for proteome-wide relative quantitation of large batches of samples3,4,5,6. Tandem mass tags (TMT)-based quantitation is a popular choice for its high multiplexing capability7,8. TMT reagents were initially launched as a 6-plex kit capable of quantifying up to 6 samples simultaneously9. This technology was further expanded to quantify 10-11 samples10,11. Recently developed 16-plex TMTpro (termed TMT16 hereafter) reagents have further increased the multiplexing capacity to 16 samples in a single experiment12,13. The TMT16 reagents use a proline-based reporter group, whereas 11-plex TMT applies a dimethylpiperidine-derived reporter group. Both TMT11 and TMT16 use the same amine reactive group, but the mass balance group of TMT16 is larger than that of TMT11, enabling the combination of 8 stable C13 and N15 isotopes in the reporter ions to achieve 16 reporters (Figure 1).
The increase in multiplexing capability provides a platform for designing experiments with sufficient replicates to overcome statistical challenges14. Furthermore, the additional channels in the 16-plex TMT help reduce the total amount of starting material per channel, which may aid in the development of emerging single-cell proteomics15. The high multiplexing capacity will also be valuable in quantitation of post-translational modifications, which typically requires high amounts of starting material16,17.
Proteomic workflows employing TMT technology have been streamlined18,19,20, and they have evolved significantly over the past decade in terms of sample preparation, liquid chromatography separation, mass spectrometric data acquisition, and computational analysis21,22,23,24,25,26. Our previous article provides an in-depth overview of the 10-plex TMT platform27. The protocol described here introduces a detailed, optimized method for TMT16, including protein extraction and digestion, TMT16 labeling, sample pooling and desalting, basic pH, and acidic pH reverse phase (RP) LC, high-resolution MS, and data processing (Figure 2). The protocol also highlights the key quality control steps that have been incorporated for successfully completing a quantitative proteomics experiment. This protocol can be routinely used to identify and quantify greater than 10,000 proteins with high reproducibility, to study biological pathways, cellular processes, and disease progression20,28,29,30.

<table>
	<tbody>
		<tr>
			<td>10% Criterion TGX Precast Midi Protein Gel</td>
			<td>Biorad</td>
			<td>5671035</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TGS (Tris/Glycine/SDS) Buffer</td>
			<td>BioRad</td>
			<td>161-0772</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8211;20% Criterion TGX Precast Midi Protein Gel</td>
			<td>Biorad</td>
			<td>5671095</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Hydroxylamine</td>
			<td>Thermo Scientific</td>
			<td>90115</td>
			<td></td>
		</tr>
		<tr>
			<td>6 X SDS Sample Loading Buffer</td>
			<td>Boston Bioproducts Inc</td>
			<td>BP-111R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate (NH4COOH)</td>
			<td>Sigma</td>
			<td>70221-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide, 28%</td>
			<td>Sigma</td>
			<td>338818-100ml</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 Ziptips</td>
			<td>Harvard Apparatus</td>
			<td>74-4607</td>
			<td>Used for desalting</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>Gel Code Blue Stain Reagent</td>
			<td>Thermo</td>
			<td>24592</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>HPLC Grade Acetonitrile</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH015-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade Water</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH365-4</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>Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>Q Exactive HF</td>
			<td></td>
		</tr>
		<tr>
			<td>MassPrep BSA Digestion Standard</td>
			<td>Waters</td>
			<td>186002329</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH230-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoflow UPLC</td>
			<td>Thermo Scientific</td>
			<td>Ultimate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</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-Pack Columns</td>
			<td>New Objective</td>
			<td>PF360-75-15-N-5</td>
			<td></td>
		</tr>
		<tr>
			<td>SepPak 1cc 50mg</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Used for desalting</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedvac</td>
			<td>Thermo Scientific</td>
			<td>SPD11V</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTpro 16plex Label Reagent Set</td>
			<td>Thermo Scientific</td>
			<td>A44520</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>Ultra-micro Spin Column,C18</td>
			<td>Harvard apparatus</td>
			<td>74-7206</td>
			<td>Used for desalting</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>Used for basic pH LC</td>
		</tr>
	</tbody>
</table>

high-throughput and deep-proteome profiling by 16-plex tandem mass tag labeling coupled with two-dimensional chromatography and mass spectrometry

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

The protocol for the newly developed TMT16, including labeling reaction, desalting, and LC-MS conditions, has been systematically optimized41. Furthermore, we directly compared the 11-plex and 16-plex methods by using them to analyze the same human AD samples41. After optimization of the key parameters for TMT16, both TMT11 and TMT16 methods yield similar proteome coverage, identification, and quantification &#62; 100,000 peptides in &#62; 10,000 human proteins.
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Read this Scientific Article Protocol about High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry at JoVE.co

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

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

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