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.

Quantification of membrane and membrane-bound proteins in normal and malignant breast cancer cells isolated from the same patient with primary breast carcinoma.

More than 50% of all major drug targets are membrane proteins, and their role in cell-cell interaction and signal transduction is a vital concern. By culturing normal and malignant breast cancer cells with light or heavy isotopes of amino acids (SILAC), followed by cell fractionation, 1D gel separation of crude membrane proteins, and analysis of the digests using nanoelectrospray LC-MS/MS, we have quantified 1600 gene products that group into 997 protein families with approximately 830 membrane or membrane-associated proteins; 100 unknown, unnamed, or hypothetical proteins; and 65 protein families classified as ribosomal, heat shock, or histone proteins. A number of proteins show increased expression levels in malignant breast cancer cells, such as autoantigen p542, osteoblast-specific factor 2 (OSF-2), 4F2 heavy chain antigen, 34 kDa nucleolar scleroderma antigen, and apoptosis inhibitor 5. The expression of other proteins, such as membrane alanine aminopeptidase (CD13), epididymal protein, macroglobulin alpha2, PZP_HUMAN, and transglutaminase C, decreased in malignant breast cancer cells, whereas the majority of proteins remained unchanged when compared to the corresponding nonmalignant samples. Downregulation of CD13 and upregulation of OSF-2 were confirmed by immunohistochemistry using human tissue arrays with breast carcinomas. Furthermore, at least half the gene products displaying an expression change of 5-fold or higher have been described previously in the literature as having an association with cancerous malignancy. These results indicate that SILAC is a powerful technique that can be extended to the discovery of membrane-bound antigens that may be used to phenotype diseased cells.

Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH.

The pH of conventional Tris-glycine SDS-PAGE gels during a run is determined to be 9.5, in contrast to Bis-Tris-Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10mM acrylamide and pH 7.2 is more than 4h at room temperature. Rates are significantly lower in Bis-Tris-Mes gels than in Tris-glycine gels, reducing the risk of adventitious protein modification. Commercial Bis-Tris-Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris-glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis-Tris gels.