Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.
Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.
Yuichi
Y
Abe
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.
Maiko
M
Nagano
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.
Asa
A
Tada
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.
Jun
J
Adachi
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.
Takeshi
T
Tomonaga
D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D030562:Databases, Protein; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047468:Immunoprecipitation; D048292:Mitogen-Activated Protein Kinase 13 / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D019000:Phosphotyrosine; D014443:Tyrosine; D000069347:Erlotinib Hydrochloride; D048292:Mitogen-Activated Protein Kinase 13; C512478:EGFR protein, human; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human
cancer; immunoprecipitation; kinome; phosphorylation signaling; phosphotyrosine