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.