Biology:Tandem mass tag
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][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 | |
---|---|
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 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][non-primary source needed]
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]
References
- ↑ "Compositional Proteomics: Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags". Journal of Proteome Research 17 (1): 590–599. January 2018. doi:10.1021/acs.jproteome.7b00699. PMID 29195270.
- ↑ "Multibatch TMT Reveals False Positives, Batch Effects and Missing Values". Molecular & Cellular Proteomics 18 (10): 1967–1980. October 2019. doi:10.1074/mcp.RA119.001472. PMID 31332098.
- ↑ "Proteome-Wide Evaluation of Two Common Protein Quantification Methods". Journal of Proteome Research 17 (5): 1934–1942. May 2018. doi:10.1021/acs.jproteome.8b00016. PMID 29635916.
- ↑ "Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides". Analytical Chemistry 91 (18): 11606–11613. September 2019. doi:10.1021/acs.analchem.9b01814. PMID 31418558.
- ↑ "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. doi:10.1021/ac0262560. PMID 12713048.
- ↑ "Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags". Analytical Chemistry 80 (8): 2921–31. April 2008. doi:10.1021/ac702422x. PMID 18312001.
- ↑ "Ion coalescence of neutron encoded TMT 10-plex reporter ions". Analytical Chemistry 86 (7): 3594–601. April 2014. doi:10.1021/ac500140s. PMID 24579773.
- ↑ "Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-Statistics with Peptide Quantification Concordance". 2017. bioRxiv 10.1101/210476.
- ↑ "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. doi:10.1007/s13361-016-1434-9. PMID 27400695. Bibcode: 2016JASMS..27.1620P.
- ↑ "Optimizing Accuracy and Depth of Protein Quantification in Experiments Using Isobaric Carriers". Journal of Proteome Research 20 (1): 880–887. January 2021. doi:10.1021/acs.jproteome.0c00675. PMID 33190502.
- ↑ "SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation". Genome Biology 19 (1): 161. October 2018. doi:10.1186/s13059-018-1547-5. PMID 30343672.
- ↑ "Single-cell protein analysis by mass spectrometry". Current Opinion in Chemical Biology 60: 1–9. February 2021. doi:10.1016/j.cbpa.2020.04.018. PMID 32599342.
![]() | Original source: https://en.wikipedia.org/wiki/Tandem mass tag.
Read more |