tandem mass tag

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;{{cite journal | vauthors = Thompson A, Schäfer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C | display-authors = 6 | title = Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS | journal = Analytical Chemistry | volume = 75 | issue = 8 | pages = 1895–904 | date = April 2003 | pmid = 12713048 | doi = 10.1021/ac0262560 }} TMTsixplex, an isobaric set of six mass tags with five isotopic substitutions;{{cite journal | vauthors = Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC | display-authors = 6 | title = Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags | journal = Analytical Chemistry | volume = 80 | issue = 8 | pages = 2921–31 | date = April 2008 | pmid = 18312001 | doi = 10.1021/ac702422x }}{{Primary source inline|date=February 2019}} 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,{{cite journal | vauthors = Werner T, Sweetman G, Savitski MF, Mathieson T, Bantscheff M, Savitski MM | title = Ion coalescence of neutron encoded TMT 10-plex reporter ions | journal = Analytical Chemistry | volume = 86 | issue = 7 | pages = 3594–601 | date = April 2014 | pmid = 24579773 | doi = 10.1021/ac500140s }}{{Primary source inline|date=February 2019}} TMTpro a 16 plex version with a different reporter and mass normalizer than the original TMT, and TMTpro Zero.

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.

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.{{cite bioRxiv|last1=Peshkin|first1=L.|last2=Ryazanova|first2=L.|last3=Wuhr|first3=M.|title=Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-Statistics with Peptide Quantification Concordance|date=2017|display-authors=etal|biorxiv=10.1101/210476}} The TKO standard can be used to assess interference {{cite journal | vauthors = Paulo JA, O'Connell JD, Gygi SP | title = A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments | journal = Journal of the American Society for Mass Spectrometry | volume = 27 | issue = 10 | pages = 1620–5 | date = October 2016 | pmid = 27400695 | pmc = 5018445 | doi = 10.1007/s13361-016-1434-9 | bibcode = 2016JASMS..27.1620P }}{{Primary source inline|date=February 2019}}

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.{{cite journal | vauthors = Specht H, Slavov N | title = Optimizing Accuracy and Depth of Protein Quantification in Experiments Using Isobaric Carriers | journal = Journal of Proteome Research | volume = 20 | issue = 1 | pages = 880–887 | date = January 2021 | pmid = 33190502 | pmc = 7775882 | doi = 10.1021/acs.jproteome.0c00675 }} Such isobarically labeled samples are referred to as isobaric carriers. They were introduced for single-cell protein analysis by mass spectrometry,{{cite journal | vauthors = Budnik B, Levy E, Harmange G, Slavov N | title = SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation | journal = Genome Biology | volume = 19 | issue = 1 | pages = 161 | date = October 2018 | pmid = 30343672 | pmc = 6196420 | doi = 10.1186/s13059-018-1547-5 | doi-access = free }} and have found many other applications.{{cite journal | vauthors = Slavov N | title = Single-cell protein analysis by mass spectrometry | journal = Current Opinion in Chemical Biology | volume = 60 | pages = 1–9 | date = February 2021 | pmid = 32599342 | doi = 10.1016/j.cbpa.2020.04.018| arxiv=2004.02069 | pmc = 7767890 }}

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{{Quantitative proteomics}}

Category:Biochemistry detection methods

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Category:Mass spectrometry