Lowering Sample Requirements to Study Tyrosine Kinase Signaling using Phosphoproteomics with the TMT Calibrator Approach

ABSTRACT: Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation (IP) of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile 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 in 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 even smaller samples. Here, we demonstrate that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling 10-fold (to ~1 mg total protein per sample from 107 cells grown in 1 plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell Of Lung

DISEASE(S): Lung Adenocarcinoma

SUBMITTER: John Koomen  

LAB HEAD: John M. Koomen, PhD

PROVIDER: PXD018952 | Pride | 2020-07-29

REPOSITORIES: Pride