Liang Xiquan, Fonnum Geir, Hajivandi Mahbod, Stene Torkel, Kjus Nini H, Ragnhildstveit Erlend, Amshey Joseph W, Predki Paul, Pope R Marshall
Invitrogen Corporation, Carlsbad, California, USA.
J Am Soc Mass Spectrom. 2007 Nov;18(11):1932-44. doi: 10.1016/j.jasms.2007.08.001. Epub 2007 Aug 14.
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.
蛋白质磷酸化调节细胞功能的许多方面，包括细胞增殖、迁移和信号转导。从大量未磷酸化肽中分离磷酸肽的有效策略对于使用质谱进行全面表征至关重要。我们描述了一种采用相对和绝对定量同位素标记试剂（iTRAQ）标记的方法，以定量比较商业和原型固定化金属亲和螯合（IMAC）及金属氧化物树脂。结果表明，与磁珠偶联的原型铁螯合树脂优于Ga(3+)偶联类似物、Fe(3+)或负载Ga(3+)的亚氨基二乙酸（IDA）包被磁颗粒、负载Ga(3+)的Captivate磁珠、负载Fe(3+)的Poros 20MC或锆包被的ProteoExtract磁珠。例如，与Poros 20MC相比，此处研究的磁性金属螯合物（MMC）使磷酸肽回收率提高了20%，且未磷酸化肽的污染减少了60%。在效率和污染方面，MMC的表现与原型磁性金属氧化物包被（TiO(2)）磁珠（MMO）或TiO(2)色谱球相当，即使后者与2,5 - 二羟基苯甲酸（DHB）程序一起使用。到目前为止，新原型的灵敏度达到50 fmol，与TiO(2)球相当。在对天然蛋白质组的探索中，用5×10(7)个HeLa细胞进行表皮生长因子（EGF）处理后，从细胞培养中氨基酸稳定同位素标记（SILAC）标记的免疫复合物中捕获的胰蛋白酶（磷酸）肽足以定量60多种蛋白质的刺激反应并鉴定20个特定的磷酸化位点。
