title: Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.
abstract: BACKGROUND
  Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics.
  
  
  METHODS
  Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled phosphotyrosine antibodies P-Tyr-1000 (N=3) and 4G10 (N=3). NanoLC-MS/MS was performed using a Q Exactive mass spectrometer. For relative quantitation of protein phosphorylation, spectral counts of phosphoproteins and ion intensities of phosphopeptides were determined using MaxQuant.
  
  
  RESULTS
  From the 3 samples incubated with P-Tyr-1000 a total of 689 phosphopeptides were identified with 60% ID reproducibility. The phosphopeptide capture using 4G10 resulted in a total of 421 at 46% ID reproducibility. The P-Tyr-1000 was applied to EGFR mutated U87 cells. Erlotinib reduced EGFR phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. EGFR inhibition was accompanied by enhanced phosphorylation of FYN, MET, PTK2, DYRK1A, MAPK1 and EPHA2.
  
  
  CONCLUSIONS
  The P-Tyr-1000 phosphotyrosine antibody performs superiorly when compared to 4G10 antibody for label-free phosphotyrosine-based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of tyrosine kinase inhibitors.
  
  
  CONCLUSIONS
  In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomic analyses have meanwhile emerged as a sophisticated approach for the determination of protein phosphorylation. These analyses rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than 4G10 antibody, while including almost all 4G10 captured phosphopeptides. The workflow can be reproducibly performed at intermediate protein input levels of 10mg. Furthermore, application of the P-Tyr-1000 antibody in a standardized phosphoproteomics workflow allows relative quantitation of drug target inhibition and provides insights in alternative signaling pathways in cancer cells. This article is part of a Special Issue entitled: HUPO 2014.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: van der Mijn
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Mariette
authors.initials: M
authors.lastname: Labots
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Thang V
authors.initials: TV
authors.lastname: Pham
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Jaco C
authors.initials: JC
authors.lastname: Knol
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Broxterman
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: h.verheul@vumc.nl.
authors.forename: Henk M
authors.initials: HM
authors.lastname: Verheul
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: c.jimenez@vumc.nl.
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
mesh_terms: D000912:Antibodies, Neoplasm / Q000737:chemistry*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D019000:Phosphotyrosine / Q000032:analysis* / Q000378:metabolism
chemical_list: D000912:Antibodies, Neoplasm; D010750:Phosphoproteins; D019000:Phosphotyrosine; D000069347:Erlotinib Hydrochloride; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Cancer; EGFR; Erlotinib; Phosphoproteomics; Signaling; Tyrosine