Zhao Liang, Wu Ren'an, Han Guanghui, Zhou Houjiang, Ren Lianbing, Tian Ruijun, Zou Hanfa
National Chromatographic R and A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
J Am Soc Mass Spectrom. 2008 Aug;19(8):1176-86. doi: 10.1016/j.jasms.2008.04.027. Epub 2008 Apr 26.
The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe(3)O(4)/SiO(2) core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol beta-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of alpha-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.
从蛋白水解消化产物中高选择性地捕获磷酸化肽段是通过质谱鉴定磷酸化蛋白面临的巨大挑战。在这项工作中，合成了膦酸锆修饰的磁性Fe(3)O(4)/SiO(2)核壳纳米颗粒，并成功将其应用于从复杂的蛋白质胰蛋白酶消化产物中选择性捕获磷酸化肽段，以便在进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析时能方便地处理样品。研究了磁性纳米颗粒与蛋白质的比例以及从复杂蛋白水解消化产物中捕获磷酸化肽段的孵育时间，优化后的纳米颗粒与蛋白质比例和孵育时间分别为15:1至30:1和30分钟。通过MALDI-TOF-MS特异性捕获膦酸锆修饰的磁性Fe(3)O(4)纳米颗粒，实现了0.5 fmol β-酪蛋白出色的检测限。在MALDI-TOF-MS分析中，膦酸锆修饰的磁性Fe(3)O(4)纳米颗粒对磷酸化肽段具有高度特异性，这通过从α-酪蛋白和牛血清白蛋白摩尔比为1:100的混合物复杂胰蛋白酶消化产物中选择性捕获磷酸化肽段得以证明。通过与纳升液相色谱-串联质谱（nano-LC-MS/MS）和串联质谱/串联质谱（MS/MS/MS）分析相结合，进一步将磁性纳米颗粒应用于从小鼠肝脏裂解物的胰蛋白酶消化产物中选择性捕获磷酸化肽段，共成功鉴定出194种独特的磷酸化肽段。
