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Updated: Dec 10, 2025
TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
Published on: June 8, 2020
Lowering Sample Requirements to Study Tyrosine Kinase Signaling Using Phosphoproteomics with the TMT Calibrator
Bin Fang1, Victoria Izumi1, Lily L Remsing Rix1
1Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL, 33612, USA.
Abstract:
Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile tyrosine kinase substrates. A typical protocol requests 10 mg of total protein from ≈108 cells or 50-100 mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemical labeling reduces the protein amount required for each sample, and newer approaches use a material-rich reference channel as a calibrator to trigger detection and quantification for smaller samples. Here, it is demonstrated that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling tenfold (to ≈1 mg total protein per sample from 107 cells grown in one plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. Data are available through PRIDE (PXD019764 for label-free and PXD018952 for TMT). This strategy opens more opportunities for pY profiling of large sample cohorts and samples with limited protein quantity such as immune cells, xenograft models, and human tumors.
Insights
This study introduces a tandem mass tag (TMT) calibrator method to significantly reduce sample input for phosphotyrosine (pY) profiling. This advancement enables deeper analysis of tyrosine kinase signaling in cancer research using smaller cell or tissue samples.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Therapy
Background:
- Tyrosine kinase signaling is crucial for targeted cancer therapies.
- Current phosphotyrosine (pY) profiling requires large sample amounts (10 mg protein) via immunoprecipitation and LC-MS/MS.
- High sample input limits feasibility for certain research, including rare cell types or limited tissue.
Purpose of the Study:
- To develop a more efficient method for phosphotyrosine (pY) profiling.
- To reduce the protein input required for pY proteome analysis.
- To enable pY profiling in samples with limited quantities.
Main Methods:
- Utilized a tandem mass tag (TMT) calibrator approach for sample multiplexing.
- Applied chemical labeling and a material-rich reference channel for quantification.
- Reduced sample input by tenfold compared to traditional methods.
Main Results:
- Achieved tenfold reduction in sample input for pY profiling (≈1 mg protein per sample).
- Maintained the depth of pY proteome sampling and biological relevance.
- Demonstrated feasibility for smaller sample sizes (10^7 cells).
Conclusions:
- The TMT calibrator approach significantly lowers sample input requirements for pY profiling.
- This method enhances opportunities for analyzing large cohorts and limited samples like immune cells and tumors.
- Facilitates broader application of pY proteome analysis in cancer research and beyond.
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