TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples

Abstract

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs—all with essentially no missing values across the 16 samples and no loss in quantitative integrity.

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Fig. 1: Overview of TMTpro reagents for sample multiplexing.
Fig. 2: Comparison of TMT0- and TMTpro0-labeled samples.
Fig. 3: Application of TMTpro reagents to eight human cell lines treated with Torin1.
Fig. 4: Application of TMTpro to a two-dimensional PISA assay.

Data availability

The MS data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD014369, PXD016491 and PXD016940. The list of human kinases was downloaded from UniProt on 9 June 2019 (https://www.uniprot.org/docs/pkinfam). Affinity data of 442 human kinases to staurosporine were downloaded from the database ‘The IUPHAR/BPS Guide to Pharmacology’ on 14 May 2019 (https://www.guidetopharmacology.org/).

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Acknowledgements

We thank members of the Gygi Laboratory, particularly R. Rad, at Harvard Medical School. This work was funded in part by NIH grant nos. 1R01GM132129 (to J.A.P.) and 5R01GM067945 (to S.P.G), and the Mark Foundation for Cancer Research Fellow of the Damon Runyon Cancer Research Foundation DRG 2359-19 (to J.G.V.V.).

Author information

Affiliations

Authors

Contributions

J.L. prepared the cell line samples treated with Torin1 for MS analysis, performed the data analyses, prepared the figures and wrote the manuscript. J.G.V.V. prepared and conducted the PISA experiments and prepared associated figures. L.P.V. proposed the eight cell lines, then grew and treated the lines for Torin1 experiments and performed western blotting experiments. D.K.S. developed and implemented the real-time online searching tool. E.L.H. advised on data analyses. R.V. and A.M.R. provided additional reagent characterization and advice. C.E., P.N., R.D.B. and J.C.R. further characterized the TMTpro reagents, initiated the collaboration and provided input and oversight for the project. Reagents were conceived, developed, synthesized and characterized by K.K., A.H.T. and I.P. S.P.G. oversaw the project and edited the manuscript. J.A.P. oversaw the project, performed the experiment for comparison of TMT0 and TMTpro0, ran the MS analysis, performed the data analyses and wrote the manuscript. All authors approved the manuscript.

Corresponding authors

Correspondence to Steven P. Gygi or Joao A. Paulo.

Ethics declarations

Competing interests

The TMTpro reagents were commercialized by ThermoFisher Scientific in September 2019. C.E., P.N., R.V., A.M.R., R.D.B. and J.C.R. are employees of ThermoFisher Scientific. A.H.T. and I.P. were employees of Proteome Sciences. K.K. is an employee of Proteome Sciences. S.P.G. is a member of the scientific advisory board for ThermoFisher Scientific.

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Peer review information Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–12.

Reporting Summary

Supplementary Table 1

Protein quantifications in 48 cell line samples treated with DMSO and Torin1 (RTS–SPS–MS3).

Supplementary Table 2

Protein quantifications in replicate1 of the 48 cell line samples treated with DMSO and Torin1 (hrMS2 and SPS–MS3).

Supplementary Table 3

Phosphorylation quantifications in 48 cell line samples treated with DMSO and Torin1.

Supplementary Table 4

Protein quantifications in the PISA assay (RTS–SPS–MS3).

Supplementary Table 5

Protein quantifications in the PISA assay (hrMS2).

Supplementary Table 6

Correction factors of the TMTpro reagents used in this work.

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Li, J., Van Vranken, J.G., Pontano Vaites, L. et al. TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat Methods 17, 399–404 (2020). https://doi.org/10.1038/s41592-020-0781-4

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