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.

Fig. 1|. Overview of TMTpro reagents for sample multiplexing.

a,b, Chemical structures of TMT (a) and TMTpro reagents (b). TMT and TMTpro reagents are available as set of up to 11plex and 16plex, respectively. TMT-127N, TMT-127C, TMTpro-127N and TMTpro-127C are shown here as examples. See Supplementary Fig. 1 for structures for all TMTpro reagents. c, Illustration of TMTpro16plex reporter ions.

Fig. 2|. Comparison of TMT0- and TMTpro0-labeled samples.

a, Trypsinized SH-SY5Y cell lysate was labeled with light (no heavy isotopes) TMT0 or TMTpro0, and analyzed by SPS–MS3. TMT0 has a monoisotopic modification mass of 224.1525. TMTpro0 has a monoisotopic modification mass of 295.1896. They both generate a reporter ion of a monoisotopic mass of 126.1277 by HCD. b, The number of TMT0- and TMTpro0-labeled peptides identified with increasing MS3 NCE. TMTpro labeling had an average of 10.3% fewer peptide identifications compared with TMT-labeling. c, Summed reporter ion signal-to-noise (SN) of TMT0- and TMTpro0-labeled peptides. Distributions are presented as box plots (center line, median; box limits correspond to the first and third quartiles; whiskers, 1.5× interquartile range). Numbers of peptides in each box are (from left to right): 4,656, 4,633, 4,723, 4,685, 4,187, 4,194, 4,168 and 4,220. TMTpro0-labeled peptides had a lower optimal MS3 NCE (45%) and generated higher reporter ion signal-to-noise than TMT0-labeled peptides at respective optimal MS3 NCE. d, Stability test for TMTpro0-labeled peptides. Trypsinized SH-SY5Y cell lysate was labeled with TMT0 and TMTpro0, respectively, mixed and analyzed at various time points (days 0–12) by HCD-hrMS2. The sample was stored at room temperature over the 12-d period. e, The number of TMT0- and TMTpro0-labeled peptides identified in the sample prepared in d. f, Proportion of TMTpro0-labeled peptides to TMT0-labeled peptides in the sample prepared in d.

Fig. 3|. Application of TMTpro reagents to eight human cell lines treated with Torin1.

a, Workflow for sample preparation. Eight human cell lines were treated with DMSO or Torin1 (an mTOR inhibitor) in three cell culture replicates for 12h and labeled with TMTpro16plex reagents. Samples were combined 1:1 across all 16 channels. Phosphopeptides were enriched with IMAC, and the flowthrough was fractionated into 24 fractions via bRPLC. Fractions were analyzed by RTS-SPS-MS3 to reduce analysis time. b, RTS-SPS-MS3 analysis metrics for protein data (12 of 24 fractions, A set). RTS-SPS-MS3 and protein closeout (two peptides per protein for each fraction or up to 24 total peptides) were used. MS time on each 16plex replicate was 18 h (1.1 h per proteome). Numbers of MS2 scans, MS3 scans, peptides, unique peptides and proteins in each replicate are shown. c, Overlap of quantified proteins across three replicates (12 of 24 fractions). More than 8,800 proteins were quantified per replicate. In total, 9,733 proteins were quantified and 7,751 were quantified in all three replicates. d,e, Unsupervised HCA (d) and PCA (e) of quantified proteins showed that samples were profiled without measurable bias or batch effect (n = 3 cell culture replicates). f, Downregulation of SQSTM1, MAP1LC3B2 and GABARAPL1 with Torin1 treatment across eight cell lines. RA stands for relative abundance. TMT RA is the percentage of total S/N within a 16plex (see Methods).

Fig. 4|. Application of TMTpro to a two-dimensional PISA assay.

a, Workflow of sample preparation. HCT116 cell lysate was divided into three aliquots and treated with the broad-spectrum protein kinase inhibitor staurosporine (stauro.). The experiment was carried out in quintuplicate. Each sample was subsequently divided into 12 tubes and a heat gradient was applied. An equal volume from each tube was pooled, centrifuged, digested and labeled with the first ten channels of TMT or the first 15 channels of TMTpro. Soluble protein abundance in vehicle (veh.) and staurosporine-treated samples, which represents the area under the melting curve, were used to measure the effect of staurosporine on protein thermal stability. b,c, TMTpro-labeling quantified proteins (b) and kinases (c) with no missing values for the PISA assay. d,e, Volcano plots of all quantified kinases under staurosporine treatment (TMTpro). Thermal stabilities of 32 kinases were affected by 1 μM staurosporine and 20 showed ≥20% AUC changes (log₂ ratio cutoff 0.26; d). Seventy-eight kinases were affected by 20 μM staurosporine and 41 showed ≥20% AUC changes (e). Destabilized kinases (dark green) and top five kinases showing high affinity to staurosporine among stabilized kinases (from database ‘The IUPHAR/BPS Guide to PHARMACOLOGY’) are labeled (n = 5 cell culture replicates, two-sided paired t-test, P values adjusted by the Benjamini-Hochberg method among all quantified kinases and filtered at 0.05).