Evaluation of inflammation-related signaling events covering phosphorylation and nuclear translocation of proteins based on mass spectrometry data

Publication date: Available online 13 November 2016
Source:Journal of Proteomics
Author(s): Andrea Bileck, Rupert L. Mayer, Dominique Kreutz, Tamara Weiss, Sabine Taschner-Mandl, Samuel M. Meier, Astrid Slany, Christopher Gerner
Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data.SignificanceRelevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.

Graphical abstract

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