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]
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][non-primary source needed] 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][non-primary source needed] 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 0
224.152478
TMT 2
225.155833
TMT 6/10
229.162932
TMT 11
229.169252
TMT Pro-zero
295.18959
TMT Pro
304.2071
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 peptidesedit
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][non-primary source needed]
Isobaric carrier conceptedit
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]
Referencesedit
^O'Brien JJ, O'Connell JD, Paulo JA, Thakurta S, Rose CM, Weekes MP, et al. (January 2018). "Compositional Proteomics: Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags". Journal of Proteome Research. 17 (1): 590–599. doi:10.1021/acs.jproteome.7b00699. PMC5806995. PMID 29195270.
^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. doi:10.1021/acs.jproteome.8b00016. PMC5984592. PMID 29635916.
^Tsai CF, Smith JS, Krajewski K, Zhao R, Moghieb AM, Nicora CD, et al. (September 2019). "Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides". Analytical Chemistry. 91 (18): 11606–11613. doi:10.1021/acs.analchem.9b01814. PMC7197904. PMID 31418558.
^Thompson A, Schäfer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, et al. (April 2003). "Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS". Analytical Chemistry. 75 (8): 1895–904. doi:10.1021/ac0262560. PMID 12713048.
^Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, et al. (April 2008). "Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags". Analytical Chemistry. 80 (8): 2921–31. doi:10.1021/ac702422x. PMID 18312001.
^Peshkin, L.; Ryazanova, L.; Wuhr, M.; et al. (2017). "Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-Statistics with Peptide Quantification Concordance". bioRxiv10.1101/210476.
^Paulo JA, O'Connell JD, Gygi SP (October 2016). "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. Bibcode:2016JASMS..27.1620P. doi:10.1007/s13361-016-1434-9. PMC5018445. PMID 27400695.
^Specht H, Slavov N (January 2021). "Optimizing Accuracy and Depth of Protein Quantification in Experiments Using Isobaric Carriers". Journal of Proteome Research. 20 (1): 880–887. doi:10.1021/acs.jproteome.0c00675. PMC7775882. PMID 33190502.
^Budnik B, Levy E, Harmange G, Slavov N (October 2018). "SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation". Genome Biology. 19 (1): 161. doi:10.1186/s13059-018-1547-5. PMC6196420. PMID 30343672.
^Slavov N (February 2021). "Single-cell protein analysis by mass spectrometry". Current Opinion in Chemical Biology. 60: 1–9. arXiv:2004.02069. doi:10.1016/j.cbpa.2020.04.018. PMC7767890. PMID 32599342.
April 13, 2024
tandem, mass, tandem, mass, chemical, label, that, facilitates, sample, multiplexing, mass, spectrometry, based, quantification, identification, biological, macromolecules, such, proteins, peptides, nucleic, acids, belongs, family, reagents, referred, isobaric. 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 Contents 1 Versions 2 Quantification of labeled peptides 3 Isobaric carrier concept 4 ReferencesVersions editThere 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 non primary source needed 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 non primary source needed 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 shiftTMT 0 224 152478TMT 2 225 155833TMT 6 10 229 162932TMT 11 229 169252TMT Pro zero 295 18959TMT Pro 304 2071The 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 editThe 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 non primary source needed Isobaric carrier concept editTMT 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 References edit O Brien JJ O Connell JD Paulo JA Thakurta S Rose CM Weekes MP et al January 2018 Compositional Proteomics Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags Journal of Proteome Research 17 1 590 599 doi 10 1021 acs jproteome 7b00699 PMC 5806995 PMID 29195270 Brenes A Hukelmann J Bensaddek D Lamond AI October 2019 Multibatch TMT Reveals False Positives Batch Effects and Missing Values Molecular amp Cellular Proteomics 18 10 1967 1980 doi 10 1074 mcp RA119 001472 PMC 6773557 PMID 31332098 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 doi 10 1021 acs jproteome 8b00016 PMC 5984592 PMID 29635916 Tsai CF Smith JS Krajewski K Zhao R Moghieb AM Nicora CD et al September 2019 Tandem Mass Tag Labeling Facilitates Reversed Phase Liquid Chromatography Mass Spectrometry Analysis of Hydrophilic Phosphopeptides Analytical Chemistry 91 18 11606 11613 doi 10 1021 acs analchem 9b01814 PMC 7197904 PMID 31418558 Thompson A Schafer J Kuhn K Kienle S Schwarz J Schmidt G et al April 2003 Tandem mass tags a novel quantification strategy for comparative analysis of complex protein mixtures by MS MS Analytical Chemistry 75 8 1895 904 doi 10 1021 ac0262560 PMID 12713048 Dayon L Hainard A Licker V Turck N Kuhn K Hochstrasser DF et al April 2008 Relative quantification of proteins in human cerebrospinal fluids by MS MS using 6 plex isobaric tags Analytical Chemistry 80 8 2921 31 doi 10 1021 ac702422x PMID 18312001 Werner T Sweetman G Savitski MF Mathieson T Bantscheff M Savitski MM April 2014 Ion coalescence of neutron encoded TMT 10 plex reporter ions Analytical Chemistry 86 7 3594 601 doi 10 1021 ac500140s PMID 24579773 Peshkin L Ryazanova L Wuhr M et al 2017 Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion Statistics with Peptide Quantification Concordance bioRxiv 10 1101 210476 Paulo JA O Connell JD Gygi SP October 2016 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 Bibcode 2016JASMS 27 1620P doi 10 1007 s13361 016 1434 9 PMC 5018445 PMID 27400695 Specht H Slavov N January 2021 Optimizing Accuracy and Depth of Protein Quantification in Experiments Using Isobaric Carriers Journal of Proteome Research 20 1 880 887 doi 10 1021 acs jproteome 0c00675 PMC 7775882 PMID 33190502 Budnik B Levy E Harmange G Slavov N October 2018 SCoPE MS mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation Genome Biology 19 1 161 doi 10 1186 s13059 018 1547 5 PMC 6196420 PMID 30343672 Slavov N February 2021 Single cell protein analysis by mass spectrometry Current Opinion in Chemical Biology 60 1 9 arXiv 2004 02069 doi 10 1016 j cbpa 2020 04 018 PMC 7767890 PMID 32599342 Retrieved from https en wikipedia org w index php title Tandem mass tag amp oldid 1215921539, wikipedia, wiki, book, books, library,
