Tandem mass tag explained

A tandem mass tag (TMT) is a chemical label that facilitates sample multiplexing in mass spectrometry (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. The relative ratio of the measured reporter ions represents the relative abundance of the tagged molecule, although ion suppression has a detrimental effect on accuracy.[1] [2] Despite these complications, TMT-based proteomics has been shown to afford higher precision than Label-free quantification.[3] In addition to aiding in protein quantification, TMT tags can also increase the detection sensitivity of certain highly hydrophilic analytes, such as phosphopeptides, in RPLC-MS analyses.[4]

Versions

There are currently six varieties of TMT available: TMTzero, a non-isotopically substituted core structure; TMTduplex, an isobaric pair of mass tags with a single isotopic substitution;[5] TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions;[6] TMT 10-plex – a set of 10 isotopic mass tags which use the TMTsixplex reporter region, but use different elemental isotope to create a mass difference of 0.0063 Da,[7] TMTpro a 16 plex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero.

Mass Shift of Different TMT Reagents!!Mass shift
TMT 0224.152478
TMT 2225.155833
TMT 6/10229.162932
TMT 11229.169252
TMT Pro-zero295.18959
TMT Pro304.2071
The tags contain four regions, namely a mass reporter region (M), a cleavable linker region (F), a mass normalization region (N) and a protein reactive group (R). The chemical structures of all the tags are identical but each contains isotopes substituted at various positions, such that the mass reporter and mass normalization regions have different molecular masses in each tag. The combined M-F-N-R regions of the tags have the same total molecular weights and structure so that during chromatographic or electrophoretic separation and in single MS mode, molecules labelled with different tags are indistinguishable. Upon fragmentation in MS/MS mode, sequence information is obtained from fragmentation of the peptide back bone and quantification data are simultaneously obtained from fragmentation of the tags, giving rise to mass reporter ions.

Quantification of labeled peptides

The structures of TMT tags are publicly available through the unimod database at unimod.org and hence, mass spectrometry software such as Mascot are able to account for the tag masses. Additionally, as of version 2.2, Mascot has the capability to quantify using TMT and other isobaric mass tags without the use of additional software. Intuitively, the trust associated with a protein measurement depends on the similarity of ratios from different peptides and the signal level of these measurements. A mathematically rigorous approach called BACIQ, that integrates peptide intensities and peptide-measurement agreement into confidence intervals for protein ratios has emerged.[8] The TKO standard can be used to assess interference [9]

Isobaric carrier concept

TMT tags are commonly used to label samples of equal abundance. If one of the labeled samples is more abundant, however, it may increase the sensitivity of the analysis for all samples.[10] Such isobarically labeled samples are referred to as isobaric carriers. They were introduced for single-cell protein analysis by mass spectrometry,[11] and have found many other applications.[12]

Notes and References

  1. O'Brien JJ, O'Connell JD, Paulo JA, Thakurta S, Rose CM, Weekes MP, Huttlin EL, Gygi SP . 6 . Compositional Proteomics: Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags . Journal of Proteome Research . 17 . 1 . 590–599 . January 2018 . 29195270 . 5806995 . 10.1021/acs.jproteome.7b00699 .
  2. Brenes A, Hukelmann J, Bensaddek D, Lamond AI . Multibatch TMT Reveals False Positives, Batch Effects and Missing Values . Molecular & Cellular Proteomics . 18 . 10 . 1967–1980 . October 2019 . 31332098 . 6773557 . 10.1074/mcp.RA119.001472 . free .
  3. O'Connell JD, Paulo JA, O'Brien JJ, Gygi SP . Proteome-Wide Evaluation of Two Common Protein Quantification Methods . Journal of Proteome Research . 17 . 5 . 1934–1942 . May 2018 . 29635916 . 5984592 . 10.1021/acs.jproteome.8b00016 .
  4. Tsai CF, Smith JS, Krajewski K, Zhao R, Moghieb AM, Nicora CD, Xiong X, Moore RJ, Liu T, Smith RD, Jacobs JM, Rajagopal S, Shi T . 6 . Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides . Analytical Chemistry . 91 . 18 . 11606–11613 . September 2019 . 31418558 . 7197904 . 10.1021/acs.analchem.9b01814 .
  5. Thompson A, Schäfer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C . 6 . Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS . Analytical Chemistry . 75 . 8 . 1895–904 . April 2003 . 12713048 . 10.1021/ac0262560 .
  6. Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC . 6 . Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags . Analytical Chemistry . 80 . 8 . 2921–31 . April 2008 . 18312001 . 10.1021/ac702422x .
  7. Werner T, Sweetman G, Savitski MF, Mathieson T, Bantscheff M, Savitski MM . Ion coalescence of neutron encoded TMT 10-plex reporter ions . Analytical Chemistry . 86 . 7 . 3594–601 . April 2014 . 24579773 . 10.1021/ac500140s .
  8. Peshkin. L.. Ryazanova. L.. Wuhr. M.. Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-Statistics with Peptide Quantification Concordance. 2017. etal. 10.1101/210476.
  9. Paulo JA, O'Connell JD, Gygi SP . A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments . Journal of the American Society for Mass Spectrometry . 27 . 10 . 1620–5 . October 2016 . 27400695 . 5018445 . 10.1007/s13361-016-1434-9 . 2016JASMS..27.1620P .
  10. Specht H, Slavov N . Optimizing Accuracy and Depth of Protein Quantification in Experiments Using Isobaric Carriers . Journal of Proteome Research . 20 . 1 . 880–887 . January 2021 . 33190502 . 7775882 . 10.1021/acs.jproteome.0c00675 .
  11. Budnik B, Levy E, Harmange G, Slavov N . SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation . Genome Biology . 19 . 1 . 161 . October 2018 . 30343672 . 6196420 . 10.1186/s13059-018-1547-5 . free .
  12. Slavov N . Single-cell protein analysis by mass spectrometry . Current Opinion in Chemical Biology . 60 . 1–9 . February 2021 . 32599342 . 10.1016/j.cbpa.2020.04.018. 2004.02069 . 7767890 .

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