hide
Free keywords:
Isoelectric focusing, Immobilized pH gradient, Mass spectrometry, Peptides, Phosphopeptides, Proteome profiling, Phosphoproteome, Synaptosomes Mouse brain
Abstract:
Protein profiling enables the qualitative characterization of a proteome
of interest. Phosphorylation is a post-translational modification with
regulatory functions in a plethora of cell processes. We present an
experimental workflow for simultaneous analysis of the proteome and
phosphoproteome with no additional enrichment for
phosphoproteins/phosphopeptides. Our approach is based on isoelectric
focusing (IEF) which allows the separation of peptide mixtures on an
immobilized pH gradient (IPG) according to their isoelectric point. Due
to the negative charge of the phosphogroup, most of the phosphopeptides
migrate toward acidic pH values. Peptides and phosphopeptides are then
identified by mass spectrometry (MS) and phosphopeptide spectra are
manually checked for the assignment of phosphorylation sites. Here, we
apply this methodology to investigate synaptosome extracts from whole
mouse brain. IEF-based peptide separation is an efficient method for
peptide and phosphopeptide identification.
