Quantitative comparison of IMAC and TiO 2 surfaces used in the study of regulated, dynamic protein phosphorylation

Xiquan Liang; Geir Fonnum; Mahbod Hajivandi; Torkel Stene; Nini H. Kjus; Erlend Ragnhildstveit; Joseph W. Amshey; Paul Predki; R. Marshall Pope

doi:10.1016/j.jasms.2007.08.001

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Quantitative comparison of IMAC and TiO 2 surfaces used in the study of regulated, dynamic protein phosphorylation

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Quantitative comparison of IMAC and TiO 2 surfaces used in the study of regulated, dynamic protein phosphorylation

Xiquan Liang, Geir Fonnum, Mahbod Hajivandi, Torkel Stene, Nini H. Kjus, Erlend Ragnhildstveit, Joseph W. Amshey, Paul Predki and R. Marshall Pope

Journal of the American Society for Mass Spectrometry, Vol.18(11), pp.1932-1944

11/01/2007

DOI: 10.1016/j.jasms.2007.08.001

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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 Ga3+-coupled analog, Fe3+, or Ga3+-loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga3+-loaded Captivate beads, Fe3+-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 (TiO2) beads (MMO) or TiO2 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 TiO2 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 × 107 HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites.

Details

Title: Subtitle: Quantitative comparison of IMAC and TiO 2 surfaces used in the study of regulated, dynamic protein phosphorylation
Creators: Xiquan Liang - Allen (United States)
Geir Fonnum - Allen (United States)
Mahbod Hajivandi - Allen (United States)
Torkel Stene - Allen (United States)
Nini H. Kjus - Allen (United States)
Erlend Ragnhildstveit - Allen (United States)
Joseph W. Amshey - Allen (United States)
Paul Predki - Allen (United States)
R. Marshall Pope - Allen (United States)
Resource Type: Journal article
Publication Details: Journal of the American Society for Mass Spectrometry, Vol.18(11), pp.1932-1944
DOI: 10.1016/j.jasms.2007.08.001
ISSN: 1044-0305
eISSN: 1879-1123
Language: English
Date published: 11/01/2007
Academic Unit: Medicine Administration
Record Identifier: 9984627194202771

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