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) identical masses (isobaric), but vary in terms of distribution of heavy isotopes around their structure. These tags- commonly referred to as tandem mass tags are designed so that the mass tag is cleaved at a specific linker region upon high-energy CID (HC), during tandem mass spectrometry yielding reporter ions of different masses.The most common isobaric tags are amine-reactive tags, but tags that react with cysteine residues and carbonyl groups have been described.[1] These amine-reactive groups go through N-hydroxysuccinimide (NHS) reactions, which are based around three types of functional groups.[1]

In a typical bottom-up proteomics workflow, protein samples are enzymatically digested by a protease to produce peptides. Each digested experimental sample is derivative with a different isotopic variant of the tag from a set, and the samples are mixed in typically equal ratios and analyzed simultaneously in one MS run. Since the tags are isobaric and have identical chemical properties, the isotopic variants of the tags appear as a single composite peak at the same m/z value in an MS1 scan with identical liquid chromatography (LC) retention times. During a liquid chromatography-mass spectrometry analysis, the fragmented peptides produce sequence-specific product ions, which is used 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. Because MS/MS is required to detect the tags, unlabeled peptides are not quantitated.

A key benefit of isobaric labeling over other quantification techniques (e.g. label-free) is the multiplex capabilities and thus increased throughput potential. The ability to combine and analyze several samples simultaneously in one LC-MS run eliminates the need to analyze multiple data sets and eliminates run-to-run variation. Without multiplexing, information can be missed from run-to-run, affecting identification and quantification, as peptides selected for fragmentation on one LC-MS/MS run may not be present or of suitable quantity in subsequent sample runs. Multiplexing reduces sample processing variability, improves specificity by quantifying the peptides from each condition simultaneously, and reduces turnaround time for multiple samples. The current available isobaric chemical tags facilitate the simultaneous analysis of 2 to 11 experimental samples.

Isobaric labeling has been successfully used for many biological applications including protein identification and quantification, protein expression profiling of normal vs abnormal states, quantitative analysis of proteins for which no antibodies are available and identification and quantification of post translationally modified proteins.

Availability

There are two types of isobaric tags commercially available: tandem mass tags (TMT)[2] and isobaric tags for relative and absolute quantitation (iTRAQ).[3] Amine-reactive TMT are available in duplex, 6-plex[4] and 10-plex and now 11-plex sets. Amine-reactive iTRAQ are available in 4-plex and 8-plex[5] forms. Sulfhydryl-reactive mass tags (iodoTMT) are available in 6-plex forms and carbonyl-reactive mass tags (aminoxyTMT) are available in 6-plex forms.

See also

References

  1. 1 2 Yates, John (2014). "Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics". ACS Publications. Retrieved February 5, 2017.
  2. ↑ 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.
  3. ↑ 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.
  4. ↑ 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.
  5. ↑ 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

  • Casado-Vela, Juan (2010). "iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range". Proteomics. 10 (2): 343–347. doi:10.1002/pmic.200900509. PMID 20029838.
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