Evaluation of different XXXX antibodies for label-free phosphoproteomics.

@entity1 @entity470 @entity5 @entity10393 @entity3963 @entity7984 @entity54748 @entity3818 @entity22129 @entity4149 @entity3866
BACKGROUND: Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of @entity4149 phosphorylation on proteins and @entity4149 kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free @entity10393 -based phosphoproteomics. METHODS: Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled @entity10393 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 @entity470 mutated U87 cells. @entity3866 reduced @entity470 phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. @entity470 inhibition was accompanied by enhanced phosphorylation of @entity7984 , MET, @entity3818 , @entity54748 , @entity3963 and @entity22129 . CONCLUSION: The P-Tyr-1000 @entity10393 antibody performs superiorly when compared to 4G10 antibody for label-free @entity10393 -based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of @entity4149 kinase inhibitors. CLINICAL SIGNIFICANCE: In the past decade multiple @entity4149 kinase inhibitors (TKIs) have been implemented in standard treatment regimens for @entity1 with @entity5 . Unfortunately the majority of @entity1 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 @entity4149 -phosphorylated peptides. Here we compared two commercially available @entity10393 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 @entity5 cells. This article is part of a Special Issue entitled: HUPO 2014.
