High throughput proteomics using isobaric tags

Proteomics techniques are evolving to become a highly sensitive, quantitative, and high-throughput approach to analyzing global protein dynamics within a cell, tissue or an organism. In the past decade, critical advances have been made across the entire proteomics workflow, most notably the development of the novel TMT10plex isobaric mass tag labeling combined with state-of-the-art advancements in analytical mass spectrometry, that have enabled more accurate, high-throughput quantitative proteomics.

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Overview of TMT-based quantitation

Isobaric chemical tags represent a fast, unbiased, and sensitive method to quantify proteins in a biological sample. They are used with a variety of samples including cells, tissues, and biological fluids, and enable relative quantitation simultaneously multiple conditions by mass spectrometry (MS).

TMT reagents can be used to label up to ten different peptide samples. For each sample, a unique reporter mass (i.e., TMT10 126-131Da) in the low mass region of the MS/MS spectrum is used to measure relative protein expression levels during peptide fragmentation. Protein quantitation is accomplished by comparing the intensities of the reporter ions.

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References

TMT workflow

Sample prep workflow for TMT-based quantitation

Protein extracts isolated from cells or tissues are reduced, alkylated and digested overnight. Samples are labeled with the TMT Reagents and then mixed before sample fractionation and clean-up. Labeled samples are analyzed by high resolutionOrbitrap LC-MS/MS to identify peptides and quantify reporter ion relative abundance.

A step-by-step TMT labeling procedure for intact proteins as well as peptides including preparing whole cell protein extracts, protein digestion, and peptide labeling.


Sample Prep reagents for improved proteome depth, quantification and throughput

Simplifying Complexity: New MS Reagents that Improve Proteome Depth, Quantification, and Throughput

This webinar focuses on an optimized proteomics sample preparation workflow for isobaric tags and highlights new tools for fractionation, sample throughput, accurate quantitation.

This 84-page handbook provides helpful hints and troubleshooting for preparation of samples from a variety of sources for MS analysis.

Download all technical handbooks ›

Enhance and Detect Lower Abundant Proteins

A step-by-step procedure for proteomics sample preparation is outlined – for abundant protein depletion and sample preparation prior to LC/MS analysis.


Additional resources

Mass spectrometry for TMT-based quantitation

TMT-based labeling combined with ultra-high resolution accurate mass (UHRAM) mass spectrometry (MS) enables greater multiplexing capacity, resulting in increased depth of quantitative proteomic analysis across larger numbers of samples. With the introduction of the TMT10plex Isobaric Mass Tag Labeling Kit, Thermo Fisher Scientific greatly increased the multiplexing potential of these experiments, namely making it possible to compare up to ten samples in a single run. The key benefit of TMT-based quantitation is that multiple samples, of any origin, can be quantified in a single analysis.

For enhanced accuracy and precision of quantitation, download this detailed workflow for running an optimized TMT experiment  .

New TMT SPS MS3 workflow

The most accurate TMT quantification of high dynamic range complex mixtures can only be accomplished by employing the Synchronous Precursor Selection (SPS) MS3 method on the Thermo Scientific™ Tribrid™ mass spectrometer family, including the Orbitrap™ Fusion™ and Orbitrap Fusion Lumos™ instruments. Four leading researchers discuss the use of tandem mass tag (TMT) multiplexing enabled by synchronous precursor selection (SPS) MS3, and how it has facilitated groundbreaking discoveries in their research.

This whitepaper, titled High Throughput Quantitative Proteomics Using Isobaric Tags, discusses critical advances driving a revolution in proteomic analysis, allowing biologists to explore the proteome at greater depth, measure changes in protein localization and quantify minute changes in low abundance proteins.


Pushing the limits of quantitation and protein characterization

The newest addition to the pioneering Tribrid line of mass spectrometers, the Orbitrap Fusion Lumos Tribrid MS expands the reach of systems biology researchers pushing the limits of quantitation and protein characterization.


Additional resources

Data analysis workflow for TMT-based quantitation

Thermo Scientific™ Proteome Discoverer 2.1 software simplifies a wide range of proteomics workflows and is recommended for quantitative analysis of TMT-based data. The software has improved data processing capabilities for handling large data sets and is enabled with study management tools, for example, to work with TMT 10-plex data sets. Version 2.1 features a new and improved TMT quantification workflow, which includes the following:

  • A new user interface for adding reporter ion isotopic distributions
  • Ability to account for reporter ion isotopic impurities for TMT10plex reagents
  • Implementation of TMT quantification based on S/N values
  • Protein and peptide quantification based on the sum of S/N values
  • New methods for normalization and scaling for peptide and protein abundance values

A demo version of Proteome Discoverer software is available free of charge. To download a free 60-day trial of Proteome Discoverer software, version 2.1, please visit the Thermo Scientific Proteomics Software Portal.


Improved Reproducibility and Protein Coverage with Proteome Discoverer 2.1

This presentation reviews the TMT-based reporter-ion quantification steps that are associated with obtaining reproducible protein quantification results using MS2 and MS3-based quantification methods.

   Presentation slides ›

   Presentation video ›

Additional resources

Sample prep workflow for TMT-based quantitation

Protein extracts isolated from cells or tissues are reduced, alkylated and digested overnight. Samples are labeled with the TMT Reagents and then mixed before sample fractionation and clean-up. Labeled samples are analyzed by high resolutionOrbitrap LC-MS/MS to identify peptides and quantify reporter ion relative abundance.

A step-by-step TMT labeling procedure for intact proteins as well as peptides including preparing whole cell protein extracts, protein digestion, and peptide labeling.


Sample Prep reagents for improved proteome depth, quantification and throughput

Simplifying Complexity: New MS Reagents that Improve Proteome Depth, Quantification, and Throughput

This webinar focuses on an optimized proteomics sample preparation workflow for isobaric tags and highlights new tools for fractionation, sample throughput, accurate quantitation.

This 84-page handbook provides helpful hints and troubleshooting for preparation of samples from a variety of sources for MS analysis.

Download all technical handbooks ›

Enhance and Detect Lower Abundant Proteins

A step-by-step procedure for proteomics sample preparation is outlined – for abundant protein depletion and sample preparation prior to LC/MS analysis.


Additional resources

Mass spectrometry for TMT-based quantitation

TMT-based labeling combined with ultra-high resolution accurate mass (UHRAM) mass spectrometry (MS) enables greater multiplexing capacity, resulting in increased depth of quantitative proteomic analysis across larger numbers of samples. With the introduction of the TMT10plex Isobaric Mass Tag Labeling Kit, Thermo Fisher Scientific greatly increased the multiplexing potential of these experiments, namely making it possible to compare up to ten samples in a single run. The key benefit of TMT-based quantitation is that multiple samples, of any origin, can be quantified in a single analysis.

For enhanced accuracy and precision of quantitation, download this detailed workflow for running an optimized TMT experiment  .

New TMT SPS MS3 workflow

The most accurate TMT quantification of high dynamic range complex mixtures can only be accomplished by employing the Synchronous Precursor Selection (SPS) MS3 method on the Thermo Scientific™ Tribrid™ mass spectrometer family, including the Orbitrap™ Fusion™ and Orbitrap Fusion Lumos™ instruments. Four leading researchers discuss the use of tandem mass tag (TMT) multiplexing enabled by synchronous precursor selection (SPS) MS3, and how it has facilitated groundbreaking discoveries in their research.

This whitepaper, titled High Throughput Quantitative Proteomics Using Isobaric Tags, discusses critical advances driving a revolution in proteomic analysis, allowing biologists to explore the proteome at greater depth, measure changes in protein localization and quantify minute changes in low abundance proteins.


Pushing the limits of quantitation and protein characterization

The newest addition to the pioneering Tribrid line of mass spectrometers, the Orbitrap Fusion Lumos Tribrid MS expands the reach of systems biology researchers pushing the limits of quantitation and protein characterization.


Additional resources

Data analysis workflow for TMT-based quantitation

Thermo Scientific™ Proteome Discoverer 2.1 software simplifies a wide range of proteomics workflows and is recommended for quantitative analysis of TMT-based data. The software has improved data processing capabilities for handling large data sets and is enabled with study management tools, for example, to work with TMT 10-plex data sets. Version 2.1 features a new and improved TMT quantification workflow, which includes the following:

  • A new user interface for adding reporter ion isotopic distributions
  • Ability to account for reporter ion isotopic impurities for TMT10plex reagents
  • Implementation of TMT quantification based on S/N values
  • Protein and peptide quantification based on the sum of S/N values
  • New methods for normalization and scaling for peptide and protein abundance values

A demo version of Proteome Discoverer software is available free of charge. To download a free 60-day trial of Proteome Discoverer software, version 2.1, please visit the Thermo Scientific Proteomics Software Portal.


Improved Reproducibility and Protein Coverage with Proteome Discoverer 2.1

This presentation reviews the TMT-based reporter-ion quantification steps that are associated with obtaining reproducible protein quantification results using MS2 and MS3-based quantification methods.

   Presentation slides ›

   Presentation video ›

Additional resources


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