Tandem Mass Tag (TMT) Multiplexing Approach to Protein Quantitation: Q&A

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Tandem Mass Tag (TMT) Multiplexing Approach to Protein Quantitation: Q&A

Team TFS
Team TFS

Proteomes and other biological samples can be difficult to analyze. That’s because proteomic samples vary in complexity from individual proteins to whole proteomes from biological systems. Increasing levels of sample complexity make identification and quantification by liquid chromatography-mass spectrometry (LC-MS) a complex, timely, and costly endeavor.


Why is running complex proteomic samples with LC-MS so challenging?

Multiplexing with TMT 2.jpgLC-MS sensitivity and throughput are a challenge when analyzing such diverse and complex samples. When running these samples through LC-MS alone, you run the risk of limiting the full-proteome interrogation of biological assays. This can lead to a misunderstanding of targeted proteins and their biological functions.


Is there another option to LC-MS alone?

Yes. Sample multiplexing using Tandem Mass Tag™ (TMT™) isobaric labeling technology, when added to your LC-MS workflow, delivers an in-depth analysis of proteomes and reveals new insights about cellular processes and pathways. For that reason, TMT-based quantitative proteomics has the potential to become the premier biological assay – and can lead labs into the future of biological study.


What is a tandem mass tag (TMT)?

It’s a chemical label that facilitates sample multiplexing in MS-based quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids. TMT belongs to a family of reagents referred to as isobaric mass tags, which are a set of molecules with the same mass but yield reporter ions of differing mass after fragmentation.


What are some advantages of TMT-based proteomics?

In some studies, TMT-based proteomics has been shown to afford higher precision than label-free quantification.1 In addition to aiding in protein quantification, TMT tags can also increase the detection sensitivity of certain highly hydrophilic analytes, such as phosphopeptides in reverse phase (RP) LC-MS analyses. Another advantage of multiplexing is increased sample throughput — combining up to 18 conditions into one experiment — which reduces instrument time and cost per sample. Quantitation based on the principle of stable isotope dilution theory also provides unmatched precision.


For example, using Thermo Scientific™ TMTpro™ 18-plex reagents for Single Cell Proteomics by Mass Spectrometry (SCoPE-MS) method, we can increase the throughput up to eight-fold, compared to label-free quantitation methods, all while improving detection and precision.


What is sample multiplexing?

Sample multiplexing is the process of analyzing large numbers of samples through a single LC-MS run by the process of pooling labeled samples and detecting the compounds’ presence and abundance by the reporter ion detection. Multiplexing experiments are closed systems that allow for more confidence in comparative results. Sample multiplexing streamlines multiple experimental models to increase biological insights.


Why is sample multiplexing important?

One important reason to use sample multiplexing is that it facilitates the complex experimental designs that better reflect and capture the nuances of complex and dynamic biological systems. Multiplexing streamlines use of multiple experimental models to increase biological insight such as biological replicates, positive and negative controls, dose-response, time series data, and rescue experiments. Because quantitation occurs for each quantifiable protein for each sample and in each experimental condition, there are essentially no missing values in the data sets obtained. In other words, because every measurement is made across every sample and treatment, TMT multiplexing experiments are closed systems with unique statistical properties that allow for more confidence in comparative results.


How does TMT multiplexing improve proteomics?

TMT multiplexing experiments are closed systems with unique statistical properties that allow for more confidence in comparative, quantitative results. This can be a powerful strategy for performing proteome-wide biological assays with increased efficiency, depth, and coverage.


Why is an HRAM instrument needed?


High-resolution accurate mass (HRAM) enabled instrumentation provides the highest possible separation of the m/z peaks. When reporter ions from the Thermo Scientific™ TMT11plex™ or TMTpro 18plex isobaric tags are released, their m/z values are 6 mDa apart. Low-resolution MS instrumentation that cannot resolve 6 mDa difference would compromise quantitative information.  Whereas Thermo Scientific™ Orbitrap™ MS can resolve all TMT reporter ions, including TMTpro 18plex, which is impossible for many other HRAM instruments.


How can instrumentation improve TMT analysis for co-isolating species?


Co-isolating species results when TMT reporter ions in a single scan are mixed with intensity from multiple precursor species. This lowers the quantitative accuracy of the technique. The solution is to use the Thermo Scientific™ Orbitrap Ascend™ Tribrid MS which has the capability to use the synchronous-precursor-selection (SPS) MS3 method template with real-time searching (RTS). This method selects fragment ions that have retained the whole TMT tag and are identified on the fly to be from only one of the co-isolating precursors and will send those fragments to MS3 to release the tag for more accurate quantitation. The SPS MS3 RTS method leads to more accurate and biologically informative results.2


1 O’Connell JD, Paulo JA, O’Brien JJ, Gygi SP (May 2018). “Proteome-Wide Evaluation of Two Common Protein Quantification Methods”. Journal of Proteome Research. 17 (5): 1934–1942.

2 Fu, Q., Liu, Z., Bhawal, R. et al. (2021) “Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for l...”. Anal Bioanal Chem 413, 419–429.