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 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, 18, 1932-1944) © 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 [11][12][13], strong cation exchange chromatography [14,15], and phosphoramidate chemistry [16,17]. For example, anti-phosphotyrosine antibodies have been used successfully to enrich tyrosine phosphorylated proteins and peptides [18 -22]. Some authors report hundreds of phosphopeptides are identi...

Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

“…To quantify relative changes in specific phosphorylation sites in purified growing and retracting neurites, we used immobilized metal-ion affinity chromatography to capture phosphopeptides and a liquid chromatography-mass spectrometry platform to identify parent proteins and their sites of phosphorylation as described under "Experimental Procedures" (52)(53)(54)(55)(56). In total, 3512 phosphopeptides were identified by MS/MS from growing and retracting neurites, representing 1883 phosphoproteins and 4145 non-redundant phosphorylation sites (supplemental Table 1).…”

A Multidimensional System for Phosphopeptide Analysis Using TiO2Enrichment and Ion-exchange Chromatography with Mass Spectrometry

“…Phosphorylated peptides have to be enriched because of their low abundance in biological materials. 35 Three different TiO 2 column lengths, (10, 20, and 30 mm) were evaluated at an injection flow rate of 5 μL/min. As shown in Table 1, use of a 10 mm TiO 2 packed column resulted in the identification of eight unique phosphorylated peptides, while use of a 20 mm column resulted in the identification of nine unique phosphorylated peptides, and a 30 mm column seven.…”

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

“…As an alternative, phosphoramidate chemistry has been developed to allow coupling phosphopeptides to a dendrimer (Tao et al, 2005;Bodenmiller et al, 2007c). The different enrichment strategies, IMAC and TiO 2 (Cantin et al, 2007;Liang et al, 2007;Marcantonio et al, 2008;Wilson-Grady, Villen, & Gygi, 2008), as well as phosphoramidate chemistry (Bodenmiller et al, 2007b), provide distinct but overlapping sets of phosphopeptides. Phosphotyrosine-containing peptides could be handled differently and targeted more specifically: the availability of highly specific antibodies that recognize this phosphomotif allowed the large-scale affinity purification of pTyrcontaining proteins/peptides.…”