Tandem Mass Tag (TMT) Systems

Table 1. Comparison of features of TMT and TMTpro reagents

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The TMTpro 16-plex and 18-plex label reagents are the next generation of TMT reagents (TMT tags) that offer increased multiplex capability and sample throughput [1]. Concurrent protein mass spectrometry analysis of up to 18 samples, derived from cells, tissues, or biological fluids can be performed with accuracy and precision.

The amine-reactive TMTpro reagents are used for labeling tryptic peptides irrespective of their protein sequence or enzyme specificity. The isobaric TMTpro label reagents are similar in design to the original TMT label reagents except that they have a longer spacer region and isobutyl proline mass reporter region (Figure 1). 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 protein quantitation.

The workflow for TMTpro and original TMT label reagents is identical (Figure 2). Additional mass spectrometry reagents such as standards, buffers, antibody and sample prep kit can be purchased separately (Table 2).

TMTpro zero label reagent (serves as a ‘light’ tag) is used for method development and labelling optimization before multiplexed analysis of labeled samples. It is a suitable alternative to costly isotope-labeled compounds and allows testing and optimization of sample preparation, labeling, fractionation, and fragmentation, for peptide identification, and reporter detection.

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Table 2. Additional MS reagents.

TMT label reagents (TMT tags) enable multiplex (2-plex to 11-plex) relative quantitation of biological samples using protein mass spectrometry. The chemical structure of TMT reagents consists of a peptide-reactive labeling group, a mass reporter, and a spacer arm (mass normalizer) (Figure 3). Based on the reactive chemistry, the TMT reagents are classified into three categories—amine reactive, cysteine-reactive, and carbonyl-reactive.

• The amine-reactive reagents are explained in the TMTpro section above. The structure is shown in Figure 3(A).
• The iodoTMT reagents react specifically with reduced cysteines (Cys) in peptides and proteins, and irreversibly label free sulfhydryl groups (Figure 3(B)).
• The aminoxyTMT reagents are optimized for efficient labeling and recovery of glycans and other carbonyl-reactive proteins, and offer greater stability of the labeled product (Figure 3(C)).

The TMT 10-plex and 11-plex label reagents share an identical structure with TMTzero, TMTduplex, and TMTsixplex reagents, but contain different numbers and combinations of 13C and 15N isotopes in the mass reporter.

Figure 3. Structural design of TMT reagents. The functional regions of the TMT reagents’ structure including MS/MS fragmentation site by higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD) and CID (collision induced dissociation). Amine-reactive, cysteine-reactive, and carbonyl-reactive TMT reagents are shown in (A), (B), and (C).

The Super heavy TMT (shTMT) and shTMTpro label reagents are non-isobaric amine-reactive TMT variants and are a suitable alternative for precursor-based protein quantification [3, 4]. These TMT derivatives contain twice the number of heavy isotopes (+11 mass units) that are dispersed throughout the reporter and spacer regions (Figure 4). They are used as a mass offset for triggering peptide selection. The workflow for Super heavy TMT (shTMT) and shTMTpro label reagents is identical (Figure 5). The shTMT label reagent is used in combination with TMT- or TMTpro-labeled samples and the mass offset from the super heavy-labeled synthetic peptide serves as a trigger for the selection of the targeted peptide in the multiplexed sample. This triggered selection reduces interference/co-isolation to enable more robust quantitation.

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The AccelerOme reagents are the newest additions to the next generation tandem mass tags (TMT) to support automated sample preparation and TMT labeling on the AccelerOme Automated Sample Preparation Platform. The integrated instrument and reagent platform enables automated sample handling of up to 36 samples for the reduction, alkylation, and digestion of proteins, followed by TMT or TMTpro labeling and clean-up. The TMT or TMTpro-labeled samples are fit-for-purpose for analysis using high resolution Thermo Scientific Orbitrap LC-MS/MS instruments (Figure 6).

Figure 6. Supporting kits and workflow of AccelerOme automated sample preparation system.

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1. Li J., Vranken J.G.V., Vaites L.P., Schweppe D.K., et al. TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat Methods, 2020. 17(4): 399–404.
2. Zecha J., Satpathy S., Kanashova T., et al. TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach. Mol Cell Proteomics, 2019. 18(7):1468–1478.  
3. Ordureau A., Yu Q., Bomgarden RD., et al. Super Heavy TMTpro Labeling Reagent: An Alternative and Higher-Charge-State-Amenable Stable-Isotope-Labeled TMTpro Variant. J Proteome Res, 2021. 20(5):3009–3013.  
4. Paulo J.A. and Gygi S.P. mTMT: An Alternative, Nonisobaric, Tandem Mass Tag Allowing for Precursor-Based Quantification. Anal Chem, 2019. 91(19):12167–12172.  

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