The Thermo Scientific Tandem Mass Tag Reagents are designed to enable identification and quantitation of proteins in different samples using tandem mass spectrometry (MS). All mass tagging reagents within a set have the same nominal mass (i.e., are isobaric) and chemical structure composed of an amine-reactive NHS ester group, a spacer arm (mass normalizer), and a mass reporter (Figure 1).

The standard tandem mass tag (TMT) labeling kits and reagents enable multiplex (6-plex to 11-plex) relative quantitation using high-resolution MS for samples prepared from cells or tissues. The TMT 10plex and 11plex label reagents share an identical structure with TMTzero, TMTduplex, and TMT 6plex reagents but contain different numbers and combinations of 13C and 15N isotopes in the mass reporter.

View a list of TMT publications in peer-reviewed journals based on topic or disease

Functional regions of the TMT reagent’s chemical structure

Figure 1. Functional regions of the TMT reagent’s chemical structure, including MS/MS sites of fragmentation by HCD and ETD.

New! TMTpro 16plex Label Reagents

Thermo Scientific TMTpro 16plex label reagents are the next generation of tandem mass tags designed to increase the level of sample multiplexing without compromising on protein identification and quantitation. The new TMTpro label reagents are similar in design to TMT label reagents in that they are both isobaric and amine-reactive, but TMTpro reagents differ in structure, having a longer spacer region and isobutyl proline mass reporter region (Figure 2). After MS/MS fragmentation, each TMTpro tag generates a unique reporter mass (126–134 Da) in the low-mass region of the high-resolution MS/MS spectrum that is used for relative quantitation of protein expression levels.

The new TMTpro 16plex label reagents have the same labeling efficiency and peptide/protein ID rates and provide the same level of quantitative precision as the original TMT reagents, with the benefit of improved quantitative accuracy with larger sample sets (Featured data, Figures 4 and 5).

Functional regions of the TMTpro reagent’s chemical structure

Figure 2. Functional regions of the TMTpro reagent’s chemical structure, including MS/MS sites of fragmentation by HCD.

*Note: TMTpro and TMT 6,10 & 11 plex reagents are mutually exclusive and cannot be mixed and matched.

Featured data

TMTPro workflow
TMT reagents data analysis and quantitation

Figure 3. Procedure summary for MS experiments using TMTpro isobaric mass tagging reagents. Protein extracts isolated from cells or tissues are reduced, alkylated, and then digested using the EasyPep Mini MS Sample Prep kit or an equivalent method. Samples are then labeled with the TMTpro reagents before sample mixing, fractionation, and cleanup. Labeled samples are analyzed on a high-resolution Orbitrap LC-MS/MS mass spectrometer before data analysis to identify peptides and quantify relative abundance of reporter ions.

Venn diagrams of overlapping proteins

Figure 4. Fewer missing values among replicates. Venn diagrams of overlapping proteins quantified among 30 replicate HeLa cell digests labeled with three TMT11plex reagent sets (top) or two TMTpro 16plex reagent sets (bottom).

Unique protein and peptide identifications of HeLa cell digest labeled with TMT11plex or TMTpro reagents

Figure 5. Peptide/Protein ID rates. Unique protein and peptide identifications of HeLa cell digest labeled with TMT11plex or TMTpro reagents analyzed by LC-MS before or after high pH reversed-phase fractionation.

TMTPro workflow
TMT reagents data analysis and quantitation

Figure 3. Procedure summary for MS experiments using TMTpro isobaric mass tagging reagents. Protein extracts isolated from cells or tissues are reduced, alkylated, and then digested using the EasyPep Mini MS Sample Prep kit or an equivalent method. Samples are then labeled with the TMTpro reagents before sample mixing, fractionation, and cleanup. Labeled samples are analyzed on a high-resolution Orbitrap LC-MS/MS mass spectrometer before data analysis to identify peptides and quantify relative abundance of reporter ions.

Venn diagrams of overlapping proteins

Figure 4. Fewer missing values among replicates. Venn diagrams of overlapping proteins quantified among 30 replicate HeLa cell digests labeled with three TMT11plex reagent sets (top) or two TMTpro 16plex reagent sets (bottom).

Unique protein and peptide identifications of HeLa cell digest labeled with TMT11plex or TMTpro reagents

Figure 5. Peptide/Protein ID rates. Unique protein and peptide identifications of HeLa cell digest labeled with TMT11plex or TMTpro reagents analyzed by LC-MS before or after high pH reversed-phase fractionation.

Highlights

  • Multiplex—concurrent MS analysis of up to 16 sample derived from cells, tissues, or biological fluids
  • Robust—increased multiplex capability results in fewer missing quantitative values among samples and within replicates for better quantitative accuracy and precision
  • Efficient—amine-reactive, NHS ester–activated reagent ensure efficient labeling of all peptides regardless of protein sequence or proteolytic enzyme specificity
  • Compatible—optimized for use with high-resolution Thermo Scientific Orbitrap platforms, including the Orbitrap Eclipse Tribrid and Orbitrap Exploris 480mass spectrometers with data analysis fully supported by Thermo Scientific Proteome Discoverer 2.4 software

Choose the right TMT product for your applications

  Amine-reactive 6-plex TMT Sixplex TMT Amine-reactive 10-plex TMT 10plex TMT Amine-reactive 11-plex TMT 11plex TMT TMTpro 16plex Label Reagent Set 16plex TMTpro
Reactive chemistry Amine-reactive, cysteine-reactive and carbonyl-reactive mass tags Amine-reactive mass tags Amine-reactive mass tags Amine-reactive mass tags
Plex 6 10 11 16
Formats Labeling reagents & kits Labeling reagents & kits Labeling reagents Labeling reagents
Standard packaging 0.2, 0.8 and 5 mg/vial 0.2, 0.8, and 5 mg/vial 0.8 and 5 mg/vial 0.5 and 5 mg/vial
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Kit

Cysteine-reactive:
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Kit

Carbonyl-reactive:
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(0.2 mg)


Reagents
(0.8, 5 mg)


Kit
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(0.8, 5 mg)
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Featured video

TMT10plex reagents in action

Learn how to prepare and label peptide samples with tandem mass tags for quantitative proteomics analysis.

Want to learn more about high-throughput multiplexed protein quantitation?

Visit thermofisher.com/tmt to learn more about Tandem Mass Tag (TMT) Quantitation products and workflow solutions. Learn how quatitative proteomics works, explore featured mass spectrometry instruments and software for quatitative proteomics, read about advances in tandem mass tag MS, download free resources including ebooks and much more.

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