Proteomic Analysis of RON Isoforms

Immunoprecipitation was performed on 3 mg of protein with RON c-terminal (E9, Santa Cruz) and RON N-terminal (RON8, Imclone) then beads were submitted and sample preparation performed as previously described ^{41 (link)}. Trypsin-digested peptides were analyzed by high pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization ^{42 (link)}. The nanospray ionization experiments were performed using a TripleTof 5600 hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale reversed-phase HPLC (Tempo). The collected data were analyzed using MASCOT^® (Matrix Sciences) and Protein Pilot 4.0 (ABSCIEX) for peptide identifications. RON isoform protein sequences are not normally included in the Protein Pilot analysis software and were supplemented in order to determine their detection.

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Chakedis J., French R., Babicky M., Jaquish D., Howard H., Mose E., Lam R., Holman P., Miyamoto J., Walterscheid Z, & Lowy A.M. (2015). A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells. Oncogene, 35(25), 3249-3259.

Publication 2015

TripleTof 5600 hybrid mass spectrometer Protein Pilot 4.0

High pressure liquid chromatography Hybrid Immunoprecipitation Isoform Ms ms Peptide Protein Ron protein Trypsin

Corresponding Organization :

Other organizations : University of California, San Diego

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Variable analysis

independent variables

Antibody used for immunoprecipitation (RON c-terminal (E9, Santa Cruz) and RON N-terminal (RON8, Imclone))

dependent variables

Identification of RON isoform proteins detected in the mass spectrometry analysis

control variables

Protein concentration used for immunoprecipitation (3 mg)
Sample preparation protocol as previously described
Trypsin digestion of peptides
High pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization
Data analysis using MASCOT and Protein Pilot 4.0 software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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