title: Quantitative comparison of IMAC and TiO2 surfaces used in the study of regulated, dynamic protein phosphorylation.
abstract: Protein phosphorylation regulates many aspects of cellular function, including cell proliferation, migration, and signal transduction. An efficient strategy to isolate phosphopeptides from a pool of unphosphorylated peptides is essential to global characterization using mass spectrometry. We describe an approach employing isotope tagging reagents for relative and absolute quantification (iTRAQ) labeling to compare quantitatively commercial and prototypal immobilized metal affinity chelate (IMAC) and metal oxide resins. Results indicate a prototype iron chelate resin coupled to magnetic beads outperforms either the Ga(3+)-coupled analog, Fe(3+), or Ga(3+)-loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga(3+)-loaded Captivate beads, Fe(3+)-loaded Poros 20MC, or zirconium-coated ProteoExtract magnetic beads. For example, compared with Poros 20MC, the magnetic metal chelate (MMC) studied here improved phosphopeptide recovery by 20% and exhibited 60% less contamination from unphosphorylated peptides. With respect to efficiency and contamination, MMC performed as well as prototypal magnetic metal oxide-coated (TiO(2)) beads (MMO) or TiO(2) chromatographic spheres, even if the latter were used with 2,5-dihydroxybenzoic acid (DHB) procedures. Thus far, the sensitivity of the new prototypes reaches 50 fmol, which is comparable to TiO(2) spheres. In an exploration of natural proteomes, tryptic (phospho)peptides captured from stable isotopic labeling with amino acids in cell culture (SILAC)-labeled immunocomplexes following EGF-treatment of 5 x 10(7) HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites.
authors.affiliation: Invitrogen Corporation, Carlsbad, California, USA. xiquan.l@invitrogen.com
authors.forename: Xiquan
authors.initials: X
authors.lastname: Liang
authors.forename: Geir
authors.initials: G
authors.lastname: Fonnum
authors.forename: Mahbod
authors.initials: M
authors.lastname: Hajivandi
authors.forename: Torkel
authors.initials: T
authors.lastname: Stene
authors.forename: Nini H
authors.initials: NH
authors.lastname: Kjus
authors.forename: Erlend
authors.initials: E
authors.lastname: Ragnhildstveit
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Amshey
authors.forename: Paul
authors.initials: P
authors.lastname: Predki
authors.forename: R Marshall
authors.initials: RM
authors.lastname: Pope
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007502:Iron Chelating Agents / Q000737:chemistry; D007553:Isotope Labeling; D008280:Magnetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D013499:Surface Properties; D014025:Titanium
chemical_list: D007502:Iron Chelating Agents; D010748:Phosphopeptides; C009495:titanium dioxide; D004815:Epidermal Growth Factor; D014025:Titanium