Isobaric labeling

A schematic of isobaric labeling. Proteins are extracted from different conditions or cell types, digested into peptides, and labeled with isobaric stable isotope tags. These tags consist of reporter, balance, and reactive regions. Lighter reporter regions are paired with heavier balance regions, such that the entire tag attached to the peptide adds the same mass shift. Therefore, after mixing, in MS1, the peptides appear as a single precursor. However, when fragmented during MS2, in addition to the normal fragment ions, the reporter regions dissociate to produce ion signals which provide quantitative information regarding the relative amount of the peptide in the samples.

Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with various chemical groups that are (at least nominally) isobaric, or the same in mass, but which fragment during tandem mass spectrometry to yield reporter ions of different mass. In a typical bottom-up proteomics workflow, proteins are enzymatically digested by a protease to produce peptides, which are then labeled with different isobaric tags. The samples are mixed in equal ratios. During a liquid chromatography-mass spectrometry analysis, the peptides are fragmented to produce sequence-specific product ions, which help to determine the peptide sequence, as well as the reporter tags, whose abundances reflect the relative ratio of the peptide in the samples that were combined.

Availability

There are two types of isobaric tags commercially available: tandem mass tags (TMT)[1] and isobaric tags for relative and absolute quantitation (iTRAQ).[2] TMT is available in duplex and 6-plex[3] forms, while iTRAQ is available in 4-plex and 8-plex[4] forms.

See also

References

  1. ↑ Thompson A, Schäfer J, Kuhn K et al. (2003). "Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS". Anal. Chem. 75 (8): 1895–904. doi:10.1021/ac0262560. PMID 12713048.
  2. ↑ Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004). "Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents". Mol. Cell. Proteomics 3 (12): 1154–69. doi:10.1074/mcp.M400129-MCP200. PMID 15385600.
  3. ↑ Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC (2008). "Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags". Anal. Chem. 80 (8): 2921–31. doi:10.1021/ac702422x. PMID 18312001.
  4. ↑ Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, Guertin S, Pribil P, Lee KH (2007). "8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease". Proteomics 7 (20): 3651–60. doi:10.1002/pmic.200700316. PMC 3594777. PMID 17880003.

Further reading