Quantitative Comparison of IMAC and TiO 2 Surfaces Used in the Study of Regulated, Dynamic Protein Phosphorylation ARTICLE IN PRESS

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 3ϩ -coupled analog, Fe 3ϩ , or Ga 3ϩ -loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga 3ϩ -loaded Captivate beads, Fe 3ϩ -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 ϫ 10 7 HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites. (J Am Soc Mass Spectrom 2007, xx, xxx) © 2007 American Society for Mass Spectrometry C haracterization of protein phosphorylation status, especially following stimulated transduction events, can provide mechanistic insights into the biological basis of signaling, cell cycle progression, adhesion, migration, and numerous other functions. Nowadays, the identification of phosphorylation sites in a complex milieu is carried out mainly by mass spectrometry. However, the sensitivity of analysis is largely hindered by low stoichiometry of phosphorylation, the reversible nature of the modification, and relatively weak ionization of phosphopeptides. It has been noted that enrichment of phosphopeptides from a pool of unphosphorylated peptides dramatically improves the success frequency for characterization. Several methods for enriching phosphorylated peptides have been reported, including chemical derivatization of phospho-residues [1-5], antibody-based capture, immobilized metal affinity chromatography (IMAC) [6 -10], enrichment on metal oxide surfaces Although 32 P-radiolabeled phosphopeptides have been used to quantify capture efficiency of a few IMAC materials in MS studies, most evaluations of capture materials have been performed in a qualitative fashio