基于分子识别纳米材料的磷酸化蛋白生物标记物检测方法

In order to increase the sensivity and accuracy of detecting phosphopretin biomarkers, new core-shell molecularly recognized nano-materials with selective adsorption, selective repulsion and anti-biofoiling together are developed. The shell is porous vinyl sulfonic acid (VSA)-containing copolymers and the core is molecularly imprinted membrane immobilized on Fe3O4 nanoparticles (Fe3O4-MIP nanoparticles).The shell exclude albumin-derived peptides and other positive charged non-phosphopeptides selectively without hindering the penetration of negative charged phosphopeptides by mechanisms including size sieving, hydrophilia residues and charege distribution of the side chains. Phosphopeptides enter the core are captured with high affinity by the core via selective binding of MIP with phosphoamino acids and residues specific to trypsine (arginine or lysine) together, affinity adsorption between Fe3+ and phosphonates, and the large binding capacity of nanoparticles stimultaneously. The shell also prevente protein and platelet adhersion (anti-biofouling) , which ensures the selective repulsion of non-phosphopeptides by the shell and selective adsorption of phosphopeptides by the core. By putting selective adsorption, selective repulsion and anti-bioflouling togher, trace phosphopeptides can be enriched effectively in one step using the core-shell molecularly recognized nano-materials. The sensitivity and accuracy of LC-MS/MS are increased as the selectivity and enrichment efficiencies of trace phosphopeptides improved. Higher LC-MS/MS sensitivity and accuracy make it possible to detect trace phosphorylated proteins in different samples with low level of total proteins. Using core-shell molecularly recognized nano-material based one step phosphopeptides enrichment method and LC-MS/MS together, phosphorylations of estrogen receptor a (ERa) in MCF-7 cells treated by estradiol and PGE2, and in different breast cancer tissues with different total proteins level are detected. Selectivity, accuracy, stability, practicality and economy of the this method to detect trace phosphoproteins are also evaluated.

为提高对痕量磷酸化蛋白生物标记物的检测灵敏度和准确性，制备集特异性吸附、选择性排斥和抗生物污渍于一体的核壳结构分子识别纳米材料：壳层通过选择性排斥作用、孔径、侧链亲水性、静电作用力，选择性排斥白蛋白多肽和正电性的其他非磷酸化多肽，但负电性的磷酸化多肽可自由通过并被核心材料吸附。核心材料同时利用人工抗体与磷酸化氨基酸和酶切位点氨基酸的特异性结合力、金属氧化物与磷酸基团的亲和吸附力、纳米微球的高吸附容量以提高对磷酸化多肽的吸附特异性和富集效率。壳层同时具有抗生物污渍性能，可防止生物大分子黏附在其表面以保证选择性排斥和特异性吸附的稳定性。利用分子识别纳米材料，一步完成样品预处理，提高样品预处理的特异性和富集效率，改善LC-MS/MS检测痕量磷酸化多肽的灵敏度和准确性。然后以雌激素受体a为模式蛋白，评价该方法对总蛋白含量不同的细胞、组织样品中ERa磷酸化的检测灵敏度、准确性、稳定性、实用性。

在Fe3O4纳米颗粒表面，制备了同时含磷酸化氨基酸和胰蛋白酶酶切位点氨基酸的核壳结构多位点MIP。多位点 MIP通过特异性识别并结合磷酸化氨基酸和酶切位点氨基酸，结合Fe3+与磷酸基团的亲和吸附力，选择性分离富集磷酸化多肽。利用MIP建立一步分离富集痕量磷酸化多肽的样品预处理方法，最优样品预处理条件为：无蛋白血清中的蛋白质，加入变性剂（6M guanidine, 50mM ammonium bicarbonate,1mM DTT）中， 370C变性12h后；在2mM碘乙酰胺黑暗中室温烷基化15min；加入胰蛋白酶：蛋白质=1:50（w/w）370C酶切时24h后，将多功能纳米微球直接加入酶切后的样品溶液中，温和震荡3h，离心沉淀MIP。然后使用乙腈（ACN）/ PBS (5 mM, pH 5.5) = 2/8淋洗3次，每次15分钟，最后ACN/NH4OH (0.15M) = 2.5/7.5将多肽洗脱后检测。在最优样品预处理条件下，一步法对混合多肽溶液中各种磷酸化多肽均可有效吸附，对长肽的吸附效率低于对短肽的吸附效率。一步法的富集特异性和富集效率高于SCX + IAMC柱，与在线样品预处理phosphochip类似。利用一步法结合LC-MS/MS可准确测定痕量a-牛眼晶体蛋白（50ug）中大部分短肽，可测定500ug的a-牛眼晶体蛋白蛋白中的全部磷酸化多肽。利用一步法结合LC-MS可测定0.5mg总细胞蛋白和0.5mg总组织蛋白中的磷酸化短肽。一步法结合LC-MS法的检测效果优于SCX + IAMC柱结合LC-MS法。一步法通过提高样品分离和富集的特异性和富集效率，提高了LC-MS测定磷酸化蛋白的准确性。.除作为样品预处理材料外，MIP还可用于直接测定磷酸化多肽中。在生物干涉传感器芯片表面制备可特异性识别磷酸化多肽的MIP，然后利用非标记的生物膜干涉法(BLI)，可快速测定a-crystallin（10-500ug/mL）和ERa（0.5-1mg细胞总蛋白）中的磷酸化多肽。样品预处理方法对BLT检测结果无影响。抗体传感器检测特异性较强，只能识别特定的磷酸化多肽，而MIP传感器的兼容性更强，可识别多种磷酸化多肽，因此灵敏度高于抗体传感器。可特异性结合磷酸化多肽的MIP与非标记检测技术联合使用，为快速准确地测定不同样品中痕量磷酸化蛋白提供了新方法。