Discovery of Cell‐Permeable Inhibitors That Target the BRCT Domain of BRCA1 Protein by Using a Small‐Molecule Microarray

BRCTs are phosphoserine‐binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain‐containing, tumor‐suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50 % of hereditary breast cancers. Cell‐permeable, small‐molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray‐based platforms, we have discovered the first cell‐permeable protein–protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)₂ domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation‐induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small‐molecule inhibitor of poly‐ADP‐ribose polymerase) and Etoposide (a small‐molecule inhibitor of topoisomerase II). Unlike previously reported peptide‐based PPI inhibitors of BRCA1, our compounds are small‐molecule‐like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP‐related pathways in DNA damage and repair response, and in cancer therapy.     

纳米材料在蛋白磷酸化分析中的应用研究

蛋白质磷酸化修饰是一种重要的蛋白质翻译后修饰,在细胞代谢过程中发挥着重要作用。当蛋白质的正常磷酸化调节发生异常时,会导致癌症、糖尿病、心脏病等各种疾病的发生。因此,蛋白磷酸化分析对于疾病的早期快速诊断、药物筛选和治疗等方面具有重大的意义。由于蛋白质磷酸化过程是动态的,并且磷酸化肽段或蛋白在生物样品中的含量较低,因此高灵敏的蛋白磷酸化分析面临着巨大的挑战。该文依据在检测过程中,选择性识别或捕获磷酸化的肽段或蛋白的主要机理,综述了近几年纳米材料对磷酸化肽段的富集和信号放大作用在蛋白磷酸化分析中的研究进展,并对其未来研究方向进行了展望。     

磷酸铵盐作为基质添加物促进磷酸化肽在MALDI-MS分析中的离子化效率

本文建立了以磷酸铵盐为添加剂的基质新系统,增强了磷酸化肽在MALDI正离子模式下的离子化。系统地考察了不同的磷酸盐以及不同的盐浓度对磷酸化肽离子化效率的影响。考察了两种适合于磷酸化肽离子化的基质类型2,5-二羟基苯甲酸和2,4,6-三羟基苯乙酮。用2,5-二羟基苯甲酸作为基质时,当加入10 mM 磷酸氢二铵时,磷酸化蛋白质β-casein的磷酸肽 48FQ[pS]EEQQQTEDELQDK63的离子化效率可以增强5-8倍,当加入10 mM磷酸二氢胺时,磷酸肽的离子化效率可以增强3-4倍。用2,4,6-三羟基苯乙酮作为基质时,当加入5mM磷酸氢二铵时,磷酸化肽的离子化效率比文献报道的最有利于磷酸化肽离子化的基质体系增强了2倍。并探讨了铵根离子和磷酸根离子促进磷酸化肽在MALDI的正离子模式下离子化效率的机理。     

基于液相色谱-串联质谱技术的磷酸化蛋白质组学分析

本文运用稳定同位素双甲基化标记技术、固定相金属离子螯合层析(IMAC)技术并结合液相色谱-串联质谱法研究了仙台病毒感染引起的宿主细胞磷酸化蛋白质组变化。实验发现定量的4 289个磷酸化肽段有~20%的蛋白质磷酸化位点发生了显著变化;通路富集分析表明哺乳动物雷帕毒素靶蛋白(mTOR)通路和剪接体(Spliceosome)通路可能参与宿主细胞对病毒的应答;通过对显著变化磷酸化肽段进行基序分析,发现了9个代表性的磷酸化修饰位点基序。本研究方法对于深入解析抗病毒天然免疫信号通路提供了新线索。     

Targeted identification of phosphorylated peptides by off-line HPLC-MALDI-MS/MS using LC retention time prediction

Protein phosphorylation is a type of posttranslational modification which plays an important role in cell regulation and signal transduction. Because of its biological relevance, a considerable amount of interest has been paid to the development of efficient techniques for phosphopeptide analysis. Although advances in MS control have enabled the high-throughput discovery of proteins from limited amounts of sample, automated selection of MS/MS precursor ions based on intensity alone can significantly hamper the detection of low-abundance phosphopeptides. On the basis of the observation that the introduction of a phosphate moiety does not dramatically change peptide retention time in reverse-phase chromatography, phosphopeptide specific MS/MS fragmentation attempts based on LC retention time and m/z were evaluated using a standard protein mixture, then using in vitro phosphorylated myelin basic protein. Results indicated that the majority (98%) of phosphopeptides identified eluted within a +/- 4-min window of the predicted LC elution time. While studies presented here are primarily proof of concept in nature, data suggest that the use of LC retention time prediction could be a valuable constraint for the identification of phosphopeptides within a set of off-line LC deposited sample spots. It is expected that the development of these methods will not only permit the targeted identification of protein phosphorylation sites but also allow the in-depth analysis of the dynamic events linked to the posttranslational modification.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

纳米氧化锆沉积棉花纤维的制备及其在磷酸化多肽快速富集中的应用

采用液相沉积法在棉花纤维的表面成功沉积了纳米氧化锆颗粒,并将其装填在移液枪的吸头内,通过移液枪的抽吸实现对磷酸化多肽的萃取,萃取过程只需要2 min,方法简单、快速。该材料不仅可以从β-酪蛋白酶解物这种简单的体系中萃取出9个磷酸化多肽,还可以从物质的量比为1∶100的β-酪蛋白酶解物和牛血清白蛋白（BSA）酶解物混合物这类含有大量非磷酸化多肽的复杂样品中萃取出4种磷酸化多肽,且没有非磷酸化多肽被检出,表现出较好的萃取选择性。将该材料应用于人血清和脱脂牛奶这两种复杂实际样品的酶解物中磷酸化多肽的快速富集萃取,分别检测出5种和9种磷酸化多肽,均表现出较好的选择性。     

介孔氧化铁材料快速、高效富集分离磷酸化肽段和蛋白的新方法研究

以0.1μmol/Lβ-casein磷酸化蛋白酶解液为对象,利用在酸性条件下对PO43-能够特异性吸附的氧化铁材料为新载体,对介孔氧化铁材料富集分离磷酸化肽段的孵育液酸含量、孵育液有机溶剂含量和洗脱液选择的条件进行了优化,结果表明:室温条件下,在含有0.1%乙酸和30%乙腈的孵育液中孵育5min后,经1mol/LNH3.H2O溶液的洗脱,介孔氧化铁可有效地将磷酸化肽段从蛋白酶解液中富集分离。本方法也可以选择性地提取α-casein磷酸化蛋白,实现了简单、快速、高效的磷酸化肽段和蛋白的富集分离。同时,通过MALDI-TOF串级质谱分析,成功地完成了在优化条件下分离出的磷酸化肽段磷酸位点的鉴定。     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。