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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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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Project description:Gene-expression profiles of rat liver cirrhosis induced by diethylnitrosamine and the effect of erlotinib on liver fibrogenesis and liver cancer development Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and the third leading cause of cancer-related death. Given the lack of successful treatment options, chemoprevention in high-risk patients has been proposed as an alternative strategy. Mounting evidence supports a role for epidermal growth factor (EGF) during chronic liver disease and hepatocellular transformation. We address the hypothesis that blocking the EGF-EGF receptor (EGFR) pathway may be an effective strategy for inhibiting fibrogenesis and hepatocarcinogenesis. A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the effects of erlotinib on underlying chronic liver disease and HCC formation. The DEN-induced rat model closely resembles disease progression in humans both pathologically and molecularly. Erlotinib significantly prevented the development of HCC tumor nodules in a dose-dependent fashion. Further, erlotinib inhibited the activation of hepatic stellate cells and prevented fibrogenesis. Erlotinib also reduced hepatotoxicity and improved liver function. Finally, a gene expression signature predictive of poor survival in human cirrhosis patients was reversed in response to erlotinib. Our data demonstrate for the first time that EGFR inhibition prevents liver fibrogenesis. Further, our results suggest that erlotinib is a potentially effective HCC chemoprevention strategy through inhibition of cirrhosis progression which can be monitored at the molecular level. Keywords: Cirrhotic liver, Expression array, Illumina, Signatures, Outcome prediction Animals received humane care according to the criteria outlined in the M-bM-^@M-^\Guide for the Care and Use of Laboratory AnimalsM-bM-^@M-^] of the National Academy of Sciences. All animals were maintained in accordance with the institutional guidelines of the Massachusetts General Hospital Subcommittee on Research Animal Care. Male Wistar rats received weekly IP injections of diethylnitrosamine (DEN) at 50 mg/kg, 100mg/kg, or vehicle control (PBS) for 18 weeks. A subset of rats received either daily (5X a week) IP injections of 2 mg/kg erlotinib or vehicle control during weeks 13 - 18. In a separate study, the erlotinib dose was lowered to 0.5 mg/kg. The vehicle groups from the two studies were not significantly different so they were combined together for analysis. Rats were weighed at the end of each week. Animals were sacrificed at 9, 13 and 19 weeks after a one-week washout period to eliminate acute effects of DEN. At the time of sacrifice, the non-tumor liver tissues were collected in RNase-free tubes and snap-frozen in liquid nitrogen. Frozen tissues were stored at -80M-BM-0C until RNA extraction.

Dataset Information

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

Publications

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

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