Quantitative comparison of IMAC and TiO2 surfaces used in the study of regulated, dynamic protein phosphorylation.
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.
Liang
Xiquan
X
Invitrogen Corporation, Carlsbad, California, USA. xiquan.l@invitrogen.com
Fonnum
Geir
G
Hajivandi
Mahbod
M
Stene
Torkel
T
Kjus
Nini H
NH
Ragnhildstveit
Erlend
E
Amshey
Joseph W
JW
Predki
Paul
P
Pope
R Marshall
RM
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
D007502:Iron Chelating Agents; D010748:Phosphopeptides; C009495:titanium dioxide; D004815:Epidermal Growth Factor; D014025:Titanium