Celikbiçak Omür, Kaynar Gizem, Atakay Mehmet, Güler Ulkü, Kayili H Mehmet, Salih Bekir
Hacettepe University, Department of Chemistry, 06800 Ankara, Turkey.
Eur J Mass Spectrom (Chichester). 2013;19(3):151-62. doi: 10.1255/ejms.1228.
Several transition metal oxides, such as iron (III), nickel (II) and zirconium (IV) oxides, were examined in detail for the specific enrichment and the purification of phosphopeptides from a digested peptide mixture solution. Phosphopeptide enrichment was performed on the metal oxide particles using a peptide mixture obtained bytryptic digestion of beta-casein. The mixture of protein digests containing bovine serum albumin (BSA): beta-casein digests (100:1 mole ratio) was also used for the phosphopeptide enrichment. Furthermore, non-fat milk digest was examined as a complex biological sample. In each phosphopeptide enrichment process, phosphopeptides were specifically enriched and separated from the non-phosphopeptides. The phosphopeptides were adsorbed onto the metal oxide surface at acidic pH values between 1.0 and 2.0 and, for desorption of phosphopeptides from metal oxide particles, pH values were examined and optimized in the enrichment studies. The analysis of phosphopeptides were carried out by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid to obtain intense protonated signals and to overcome degradation of the phosphopeptides by phosphate group loss in mass spectrometric conditions. Moreover, it was demonstrated that the direct detection of phosphopeptides from the surface of the metal oxide particles was possible using MALDI-MS by mixing the phosphopeptide-adsorbed metal oxide particles with MALDI matrix solution in slurry form before the analysis. Thus, the effects of interferences arising from chemical species used in the desorption process was successfully eliminated for the fast and sensitive detection of phosphopeptides in MALDI-MS applications.
对几种过渡金属氧化物，如氧化铁（III）、氧化镍（II）和氧化锆（IV）进行了详细研究，以从消化后的肽混合物溶液中特异性富集和纯化磷酸肽。使用通过β-酪蛋白胰蛋白酶消化获得的肽混合物在金属氧化物颗粒上进行磷酸肽富集。还使用了含有牛血清白蛋白（BSA）与β-酪蛋白消化物（摩尔比100:1）的蛋白质消化物混合物进行磷酸肽富集。此外，脱脂牛奶消化物作为复杂生物样品进行了检测。在每个磷酸肽富集过程中，磷酸肽被特异性富集并与非磷酸肽分离。磷酸肽在1.0至2.0的酸性pH值下吸附到金属氧化物表面，并且在富集研究中对从金属氧化物颗粒上解吸磷酸肽的pH值进行了检测和优化。使用含有1.0%磷酸的2,5-二羟基苯甲酸基质通过基质辅助激光解吸/电离质谱（MALDI-MS）对磷酸肽进行分析，以获得强质子化信号并克服质谱条件下磷酸肽因磷酸基团丢失而降解的问题。此外，通过在分析前将吸附有磷酸肽的金属氧化物颗粒与MALDI基质溶液以浆液形式混合，证明了使用MALDI-MS可以直接从金属氧化物颗粒表面检测磷酸肽。因此，在MALDI-MS应用中，成功消除了解吸过程中所用化学物质产生的干扰影响，实现了对磷酸肽的快速灵敏检测。
