基于质谱的硒蛋白质组学研究

Selenium （Se）is an important trace nutritive element, which has been associated with many diseases and biological processes. Selenoproteins, the major form of selenium in living organisms, are proteins containing residues of selenocysteine (Sec). Sec is the 21st amino acid, and an analogue of cysteine (Cys), with even higher reduction potential. Thus the known selenoproteins are typically characterized by their oxidoreduction activities, and become important in biological antioxidant and redox studies. It has been known that selenoproteins play key roles in cancer, cardiovascular diseases, neurodegenerative diseases, etc. .Sec is encoded by the codon, UGA, which normally represents the termination of a translation. Previous studies suggested that an insertion of Sec at UGA position requires a special mRNA element, Sec insertion sequence (SECIS). SECIS has a stem-loop structure, which is located at 3’-untranslated region (UTR) of a eukaryotic mRNA. The SECIS is usually used to predict selenoproteins by algorithms, although SECIS is not highly conserved between selenoproteins, organisms, and species. Despite the importance of selenoproteins, the known selenoproteins and the knowledge of their functions beyond oxidoreduction are very limited. To date, only 3 E. Coli, 25 human, and 24mouse selenoproteins have been discovered. This is largely because the discovery of selenoproteins relying on SECIS prediction is not always successful, and the abundance of selenoproteins are extremely low. .To address these issues, we propose to develop a Sec- specific enrichment method which employs a clickable alkylating agent, and establish a new mass spectrometry-based method to characterize the selenoproteomes of both prokaryotes and eukaryotes. This method is SECIS independent, and aiming at the complete genome, which enables us to find new possible selenoproteins. The Sec-containing peptide enrichment method proposed will greatly increase the detection sensitivity, and thus helpful in getting maximal selenoprotein coverage. With this proposal, we are able to specifically enrich Sec-containing peptide, and detect selenoproteins by using highly resolved mass spectrometry with potentials to discover new selenoproteins. The experimental results will provide further support or extensions to current SECIS-based Sec insertion mechanism. Furthermore, we will study the selenoproteome under oxidative stress conditions. The identification and quantificaiton methods of selenoproteins will be of significance in any studies using selenoproteins as candidate biomarkers.

硒蛋白是指含有硒代半胱氨酸（Sec）的蛋白，Sec是第21种氨基酸，它是半胱氨酸（Cys）的类似物，同时又比Cys具有更高的氧化还原反应活性。研究发现硒蛋白和许多疾病以及生物过程都密切相关。虽然硒蛋白很重要，但已知的硒蛋白的种类和功能都很有限，这主要是由于硒蛋白丰度过低，缺乏有效的富集和检测方法；此外，当前硒蛋白的发现主要依赖预测mRNA上的Sec插入序列（SECIS）来实现，但由于SECIS不是高度保守的，所以依靠SECIS预测硒蛋白变得不可靠。为了解决上述问题，我们拟发展可点击的烷基化试剂去特异性的富集含Sec多肽，并建立不依赖于SECIS的硒蛋白质组数据库，继而可以极大的提高检测硒蛋白的灵敏度，并有望发现新的硒蛋白。基于上述技术创新，本研究拟全面检测真核生物和原核生物的硒蛋白质组及其在氧化应激状态下的变化。这些实验结果将对我们全面了解硒蛋白质及其功能有积极推动作用。

硒蛋白是指含有硒代半胱氨酸（Sec, selenocysteine）的一类蛋白质,硒代半胱氨酸具有很高的生物活性，因此硒蛋白在维持体内的氧化还原平衡方面发挥着重要功能。硒蛋白的生物合成途径非常特殊，它利用终止密码子UGA作为密码子，将Sec插入到蛋白中，其中，mRNA 3’UTR区域的一段Sec插入序列（SECIS, selenocysteine insertion sequence）在其中发挥着重要作用。目前为止，人类中有25种硒蛋白，小鼠中有24种硒蛋白被报道。这些硒蛋白与许多疾病密切相关，例如糖尿病、癌症和神经退行性疾病等。硒蛋白具有如此重要的功能，但是其性质特殊，含量低，数量少，硒代半胱氨酸和半胱氨酸性质接近，目前尚缺乏有效的分析方法，尤其是没有高通量地对硒蛋白进行分析的方法，关于硒蛋白的功能研究以及新的硒蛋白的发现进展缓慢。因此，开发有效的高通量的硒蛋白质组研究方法迫在眉睫。.针对以上现状，我们开发了一种基于质谱的硒蛋白研究方法，称之为“SecMS”（Sec-specific mass spectrometry）。这种方法利用硒代半胱氨酸比半胱氨酸具有较低的pKa值这一特性，实现了在低pH条件下对硒代半胱氨酸进行特异性衍生化进而进行富集。此外，我们还构建了不依赖于SECIS序列的硒蛋白数据库，该数据库含有最大规模的潜在硒蛋白。结合SecMS和SIS数据库，我们不仅可以大规模地对已知硒蛋白进行研究，还可以发现新的硒蛋白。.利用SecMS方法，我们发现在BV2细胞中补充Sec可以大大提高细胞中硒蛋白的含量，硒蛋白含量的升高有利于细胞抵抗活性氧含量的升高。此外，我们用SecMS的方法对小鼠中的硒蛋白进行研究，在小鼠的9个组织中鉴定到了小鼠24个已知硒蛋白中的22个，绘制了首张硒蛋白的组织特意分布图谱。我们还发现随着年龄的增大，小鼠的硒蛋白整体呈现下降趋势，且在各组织中变化有所不同。尤为重要的是，结合SIS数据库，我们在小鼠的组织中发现了至少5个新的硒蛋白，它们均具有高度可信的谱图。这是十五年来首次报道发现新的硒蛋白，也是对硒蛋白组的最大规模扩充，SecMS结合SIS数据库的方法将对于硒蛋白的研究和新的硒蛋白发现产生深远的意义。