Dataset Information

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 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 one 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

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

DISEASE(S): Lung Adenocarcinoma

SUBMITTER: John Koomen

LAB HEAD: John M. Koomen, PhD

PROVIDER: PXD019764 | Pride | 2020-07-29

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190211_PC9_10mg_DMSO_LF.raw	Raw
	20190211_PC9_10mg_DMSO_LF.rawF008811.mzid.gz	Mzid
	20190211_PC9_10mg_DMSO_LF.rawF008811.mzid_20190211_PC9_10mg_DMSO_LF.raw_F008811.MGF	Mzid
	20190211_PC9_10mg_DMSO_LF.raw_F008811.dta	Raw
	20190211_PC9_1mg_DMSO-1_LF.raw	Raw

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

Fang Bin B Izumi Victoria V Rix Lily L Remsing LLR Welsh Eric E Pike Ian I Reuther Gary W GW Haura Eric B EB Rix Uwe U Koomen John M JM

Proteomics 20200930 24

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 ≈10<sup>8</sup> cells or 50-100 mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemi ...[more]

PMID: 32865326

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