We have developed a novel approach to enhance
phosphopeptide identification in liquid chromatography/mass spectrometry (LC/MS)-based phosphoproteomics. After enrichment of
phosphopeptides with immobilized
metal affinity chromatography (
IMAC) and
titanium dioxide (TiO(2)) microcolumns, samples were coinjected with
ethylenediaminetetraacetic acid (
EDTA) into LC/MS. This procedure decreased the MS peak intensity of nonphosphorylated
peptides, but not that of
phosphopeptides, and as a result, the number of identified
phosphopeptides was increased.
EDTA appeared to have no effect on liquid chromatographic separation of phosphorylated and nonphosphorylated
peptides. Although the mechanism of the positive effect of
EDTA on identification of
phosphopeptides is unknown, and we have never observed
metal ion adduct peaks in LC/MS spectra, coinjection of
EDTA seemed to enhance
phosphopeptide recovery from the LC/MS system. This simple technique was successfully applied to the identification of
phosphopeptides in mouse brain (2938
phosphopeptides), human plasma (127 phosphopetides), and human cerebrospinal fluid (CSF) (123
phosphopeptides). We also identified nonphosphopeptides in the same samples using a two-dimensional (2D) LC/MS-based shotgun approach. The results overall indicated that 20-25% of brain
proteins were phosphorylated, while only 1-2% of
proteins in plasma and CSF were phosphorylated. These ratios were almost constant throughout the range of
protein expression levels. In addition,
EDTA-enhanced phosphoproteomics could identify low-abundance
proteins in the samples, because nonphosphoproteins corresponding to more than one-third of the identified
phosphoproteins could not be identified by 2D-LC/MS. Finally, we were able to find that the newly developed approach was very effective for the phosphoproteome analysis in
Alzheimer disease model mice brain.