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

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 mas...

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Published inNature methods Vol. 17; no. 4; pp. 399 - 404
Main Authors Li, Jiaming, Van Vranken, Jonathan G., Pontano Vaites, Laura, Schweppe, Devin K., Huttlin, Edward L., Etienne, Chris, Nandhikonda, Premchendar, Viner, Rosa, Robitaille, Aaron M., Thompson, Andrew H., Kuhn, Karsten, Pike, Ian, Bomgarden, Ryan D., Rogers, John C., Gygi, Steven P., Paulo, Joao A.
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.04.2020
Nature Publishing Group
Subjects
631/1647/2067
631/1647/296
631/45/475
Affinity labeling
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Cell Line
Cell lines
Humans
Isotope Labeling
Kinases
Labeling
Life Sciences
Methods
Peptides
Peptides - chemistry
Proline
Proteins
Proteome - chemistry
Proteomes
Proteomics
Proteomics - methods
Reagents
Staurosporine
Tandem Mass Spectrometry - methods
Thermal stability
United States
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Summary: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. A set of isobaric labeling reagents called TMTpro enables deep quantitative comparisons of proteome measurements across 16 samples.
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content type line 23
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.
Author contributions
ISSN:1548-7091
1548-7105
DOI:10.1038/s41592-020-0781-4