LAUSR

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.