Technology of the week: TMTpro

Developed by Thermo Fisher Scientific and Gygi Lab at Harvard Medical School.

TMT (Tandem Mass Tag) mulitplexed proteomics is a powerful technique for analyzing proteomes from multiple conditions. This method allows for the simultaneous quantification of multiple samples in a single-pot, providing a route to greatly increasing the efficiency and throughput of proteomic analysis.

We perform this by adding a unique, isotopically-labeled mass tag to each digested protein/peptide sample. The digested-labeled samples are pooled into one tube before separation of peptides via HPLC (hydrophobicity) followed by electrospray ionization into the mass spectrometer.

The TMT tags are activated as N-hydroxysuccinimide esters and are covalently linked by an amide bond formation with the amino groups of peptide N-termini and at lysine residues.

TMT labeling makes way for the identification and quantification of proteins in the pool originating from multiple conditions by labeling peptides with this isobaric mass tag that contains heavy isotopes.

The results are reproducible fragmentation patterns represented by shifted peaks of defined peptide spectra which are offset by known HCD monoisotopic reporter masses (TMTpro). We look for these heavy isotope shifts represented in the mass spectra and use them as ‘barcodes’ to differentiate the samples in the pool.

This analysis is very direct and can provide valuable insights into protein expression levels, post-translational modifications, cell response to perturbations and other aspects of protein function for all samples in the plex.

This is an important tool for proteomic studies in various fields, including biomedical research, drug discovery, and clinical diagnostics.

Publication: https://www.nature.com/articles/s41592-020-0781-4
18plex publication: https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00168
Product: https://www.thermofisher.com/order/catalog/product/A52045