Comprehensive Phosphoproteome Analysis of INS-1 Pancreatic Beta-Cells using Various Digestion Strategies Coupled with Liquid Chromatography–Tandem Mass Spectrometry

Type 2 diabetes results from aberrant regulation of the
phosphorylation
cascade in beta-cells. Phosphorylation in pancreatic beta-cells has
not been examined extensively, except with regard to subcellular phosphoproteomes
using mitochondria. Thus, robust, comprehensive analytical strategies
are needed to characterize the many phosphorylated proteins that exist,
because
of their low abundance, the low stoichiometry of phosphorylation,
and the dynamic regulation of phosphoproteins. In this study, we attempted
to generate data on a large-scale phosphoproteome from the INS-1 rat
pancreatic beta-cell line using linear ion trap MS/MS. To profile
the phosphoproteome in-depth, we used comprehensive phosphoproteomic
strategies, including detergent-based protein extraction (SDS and
SDC), differential sample preparation (in-gel, in-solution digestion,
and FASP), TiO2 enrichment, and MS replicate analyses (MS2-only
and multiple-stage activation). All spectra were processed and validated
by stringent multiple filtering using target and decoy databases.
We identified 2467 distinct phosphorylation sites on 1419 phosphoproteins
using 4 mg of INS-1 cell lysate in 24 LC–MS/MS runs, of which
683 (27.7%) were considered novel phosphorylation sites that have
not been characterized in human, mouse, or rat homologues. Our informatics
data constitute a rich bioinformatics resource for investigating the
function of reversible phosphorylation in pancreatic beta-cells. In
particular, novel phosphorylation sites on proteins that mediate the
pathology of type 2 diabetes, such as Pdx-1, Nkx.2, and Srebf1, will
be valuable targets in ongoing phosphoproteomics studies.