TMT quantitative proteomics provides a high-throughput, highly sensitive, and versatile approach for assessing the efficacy and pharmacological mechanisms of PROTACs. It allows for the use of multiple sample types and enables the combination of multiple modified proteomic techniques on the same sample for more accurate investigation of post-translational modifications.
In 2003, Thompson et al. first applied isobaric labeling technology for peptide identification and relative quantification. The Tandem Mass Tag (TMT) system, designed by Thermo Fisher, is used for protein identification and quantification in different samples. Each TMT labeling reagent consists of an amine-reactive NHS ester group, a spacer arm (mass balancing region), and a mass-reporting group. TMT provides up to 16 tags that can be used to label almost any peptide or protein sample. TMT has been widely used for the discovery of quantitative protein biomarkers. TMTpro-18plex, TMTpro 16plex, TMT zero, TMT duplex, TMT sixplex, TMT 10plex, and TMT 11plex reagents have the same structure and enable efficient transition from method development to multiplexed quantification.
TMT Quantitative Proteomics Technical Workflow
Sample preparation: Sample preparation is very simple. Just send frozen cell or tissue samples, and Creative Proteomics will take care of the rest. Approximately 100 μg of total protein is sufficient for each sample.
Service Advantages
ThermoFisher™ QE series™ Lumos™ mass spectrometers, compared to other instruments, provide the most accurate quantification and the highest number of protein identification results.
① High-throughput: Allows for the analysis of protein differences in up to 16 samples simultaneously. It is suitable for large-scale experimental analysis of clinical samples or multiple control/treatment groups, especially for biomarker discovery and detection analysis of the whole proteome after drug treatment.
② High sensitivity: Enables the comparison of protein relative abundance over a wide range without compromising the detection of low-abundance proteins.
③ Multiple sample types: Suitable for analyzing differences in the whole proteome levels across various sample types, including tissues, cells, blood, etc.
④ Integration of multiple modified proteomic techniques on the same sample: Can be combined with methods for enriching post-translational modifications such as phosphorylation, enabling more accurate investigation of post-translational changes.
TMT Proteomics Applications
① Quantification of protein abundance and identification of biomarkers.
② Study of protein expression level differences in organisms after drug administration.
③ Discovery of off-target effects induced by drug stimulation, and more.
TMT Proteomics in PROTAC Drug Development
① Identification or validation of disease biomarkers or drug target proteins.
② Detection of changes in protein abundance caused by drugs.
③ Identification of potential off-target proteins, and more.
Classic case
① TMT proteomics confirmed that upregulation of CDK6 is a targetable resistance mechanism in multiple myeloma against lenalidomide.
TMT-labeled proteomics revealed differences in the global proteome and phosphorylated proteome of cells after lenalidomide treatment.
TMT experimental workflow; B. High expression of CDK6 in samples from multiple myeloma patients; C. Evaluation of proteomic and phosphorylated proteomic changes in MM1.S cells treated with different drugs using TMT-based proteomics; D. Combination heatmap of protein levels displaying paired patient data and cell perturbation data.
② TMT proteomics monitoring the differential expression of the whole proteome after PROTAC treatment.
TMT proteomics monitoring the degradation efficiency of target proteins by PROTAC3 and PROTAC5.
A. HeLa cells were treated with 1 μM PROTAC3, PROTAC4, deltasonamide 1, or VHL-based PROTAC5 for 24 hours, or 1 μM PROTAC3 or PROTAC5 for 5 hours. TMT-labeled quantitative proteomics was used to determine the protein levels in the cells. Each point represents the average p-value (n=3). All identified proteins are shown, with the colored cross indicating the abundance of PDEδ under each condition. B. Pathway analysis of significantly upregulated proteins after 24 hours of treatment with PROTAC3.
Our PROTACs Proteomics Solution
Creative Proteomics offers PROTAC degradation proteomics services, which include: (1) Extensive non-targeted protein detection using DIA and TMT to analyze the degradation efficiency of target proteins and off-target effects after PROTAC treatment. (2) Targeted quantitative detection using MRM/PRM to quantify the degradation level of target proteins after PROTAC treatment. (3) AP-MS to validate the interaction between target proteins and E3 ubiquitin ligases. (4) Ubiquitin proteomics to study the ubiquitination mechanisms of target protein degradation after PROTAC treatment. (5) Western blot-based evaluation of target protein degradation and determination of dose-response relationships.
References
- Ng YLD, Ramberger E, Bohl SR, Dolnik A, Steinebach C, Conrad T, Müller S, Popp O, Kull M, Haji M, Gütschow M, Döhner H, Walther W, Keller U, Bullinger L, Mertins P, Krönke J. Proteomic profiling reveals CDK6 upregulation as a targetable resistance mechanism for lenalidomide in multiple myeloma. Nat Commun. 2022 Feb 23;13(1):1009.
- Winzker M, Friese A, Koch U, Janning P, Ziegler S, Waldmann H. Development of a PDEδ-Targeting PROTACs that Impair Lipid Metabolism. Angew Chem Int Ed Engl. 2020 Mar 27;59(14):5595-5601.
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