TMTpro16plex Isobaric Labeling Reagents: Attaining New Sample Multiplexing Heights and New Cellular Proteome Depths in Quantitative Proteomics

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here, an expanded set of 16 isobaric reagents based on a proline backbone (TMTpro) is presented. These reagents have similar characteristics to existing TMT 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/protein) per replicate with an analysis time of only 67 min per proteome examined. Finally, we modified the powerful thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 ������M staurosporine with five replicates. This new assay identified and dose-stratified the staurosporine binding to 228 cellular kinases in just one, 18-hr experiment. TMTpro reagents allow unprecedented complexity in experimental designs���������all with basically no missing values across the 16 samples and no loss in quantitative integrity.