2.9. Proteomic and phosphoproteomic analyses
Total peptides and phosphopeptides were analyzed using LTQ-Orbitrap Elite (Thermo Fisher Scientific Inc. MA, USA) coupled with a Dionex Ultimate 3000 RSLC nano system (Thermo Fisher Scientific) and a QExactive mass spectrometer (Thermo Fisher Scientific Inc.) coupled with a Dionex Ultimate 3000 RSLC nano system, respectively. Label-free quantitation was performed using Progenesis QI for proteomics software (Nonlinear Dynamics, Newcastle, UK). The samples were divided into four groups: vehicle, OSI-906, linagliptin, and OSI-906 + linagliptin. Then, the samples were subjected to separate multivariate statistical analyses of the proteomic and phosphoproteomic data. MS/MS ion searches to identify proteins were performed using MASCOT software (version 2.5.1, Matrix Science, London, UK) against the UniProtKB database (Mus musculus, 16,678 sequences,http://www.uniprot.org/). For total peptides, protein identification was performed using the following parameters: enzyme, trypsin; peptide mass tolerance, 5 ppm; fragment mass tolerance, 0.5 Da; maximum missed cleavages, 2; and variable modifications such as protein N-terminal acetylation, carbamidomethylation of cysteine, N-terminal carbamylation and oxidation of methionine. For phosphopeptides, protein identification was performed using the following parameters: enzyme, trypsin; peptide mass tolerance, 5 ppm; fragment mass tolerance, 0.05 Da; maximum missed cleavages, 2; and variable modifications such as protein N-terminal acetylation, carbamidomethylation of cysteine, N-terminal carbamylation, oxidation of methionine, phosphorylation of serine/threonine and phosphorylation of tyrosine. We used a 1% overall false discovery rate as a cutoff value to export our results from the database search. In addition, peptides that yielded a peptide ion score of greater than or equal to 30 were used for relative quantitation. Proteins or phosphopeptides with significant quantitative changes were selected according to calculations performed using Progenesis QI for proteomics and the following parameters: analysis of variance (ANOVA) P value <0.05, and fold change >1.2.