Simultaneous identification and quantification of proteins by differential ¹⁶O/¹⁸O labeling and UPLC-MS/MS applied to mouse cerebellar phosphoproteome following irradiation

Differential proteolytic ¹⁸O labeling is a cost-effective but not commonly used method in the field of quantitative proteomics based on mass spectrometry (MS). In most cases, peptide identification is performed at the MS/MS level followed by peptide quantification at the MS level. In this study, identification and quantification of ¹⁸O-labeled peptides was performed in a single step at the MS/MS level using the MASCOT 2.2 search engine, and the instrumental conditions for acquisition of ultra performance liquid chromatography electrospray MS/MS (UPLC-ESI-MS/MS) data were adapted accordingly. Using analysis of standard peptide and protein mixtures prepared by differential ¹⁶O/¹⁸O labeling, under these conditions automated MS/MS data acquisition and evaluation delivered correct data. Linearity and reproducibility of this approach indicated excellent performance. In addition, the method was applied to relative quantification of protein phosphorylation in mouse brain following treatment with ionizing radiation. The analysis led to automated quantification of 342 proteins and 174 phosphorylation sites, 24 of which were up- or down-regulated by a factor of 2 or more. The majority of these phosphorylation sites were found to be located in target sequences of known protein kinases, showing the activation of kinase-regulated signaling cascades by irradiation.