title: Ethylenediaminetetraacetic acid increases identification rate of phosphoproteomics in real biological samples.
abstract: 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.
authors.affiliation: Eisai Company, Ltd., Biomarkers and Personalized Medicine Unit, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Nakamura
authors.forename: Khin Than
authors.initials: KT
authors.lastname: Myint
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Oda
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004195:Disease Models, Animal; D004492:Edetic Acid / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010750:Phosphoproteins / Q000097:blood / Q000134:cerebrospinal fluid / Q000737:chemistry*; D010756:Phosphoric Acids / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D014025:Titanium / Q000737:chemistry
chemical_list: D010750:Phosphoproteins; D010756:Phosphoric Acids; C009495:titanium dioxide; D004492:Edetic Acid; D014025:Titanium; C030242:phosphoric acid