Integrated workflow for quantitative phosphoproteomic analysis of the selected brain structures in development of morphine dependence

Background: Phosphorylation is a key process regulating a large number of fundamental biochemical reactions in living organisms. It is known that many mechanisms of response to chronic drugs administration are regulated by phosphorylation. It can be assumed that some of the phosphorylation sites are known, but they represent only a small fraction of the regulatory phosphorylation events in this system. Therefore, it is important to investigate protein phosphorylation with high-throughput methods such as mass spectrometry, that allow for efficient global analysis. The aim of this work was to develop a robust workflow for quantitative phosphoproteomic analysis, which operates in a semi-automatic manner. Methods: The proposed approach consists of two methods of phosphopeptides enrichment (TiO2, IMAC), stable isotope methyl labeling, data-dependent mass spectrometry acquisition with simultaneous CID/ETD fragmentation, and data analysis platform based on Trans-Proteomic Pipeline. We have applied our method to analyze selected brain structures from rat involved in morphine dependence. Results: We have identified and quantified number of phosphoproteins that were up- or down-regulated as a result of morphine treatment. Finally, we have applied a three-step filtration process to emerge the most regulated candidates. In parallel, all of the regulated proteins were annotated with GO terms to follow global trends of protein regulation. Conclusions: The proposed MS-based workflow with following data analysis is efficient method for quantitative phosphoproteomic analysis: Stable isotope methyl labeling introduces tags for quantitative analysis and in the same time increases specificity of phosphopeptides enrichment. Utilization of two complementary methods for phosphopeptides enrichment (TiO2/IMAC) broadens the phosphoproteomic coverage. Simultaneous CID/ETD fragmentation increases sequencing and phosphorylation sites mapping capabilities. Data analysis with Trans-Proteomic Pipeline allows for optimization of FDR, and efficient quantitative analysis with ASAPRatio. (C) 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.