Enabling Mass Spectrometry Analysis of the Sulfoproteome

项目摘要 酪氨酸O-硫化，即将蛋白质中的磺酸基转移到酪氨酸氨基酸残基，是一种 真核细胞中广泛存在的翻译后修饰(PTM)，具有多种已知的健康功能 以及疾病，包括受体结合、病毒复制、炎症和视网膜功能。这些酶可以 催化酪氨酸硫化位于高尔基体中。这个细胞器的功能是确保 正确的蛋白质修饰发生，并将蛋白质包装成小泡，以便输出到细胞表面，或 细胞外环境。因为蛋白质通常必须进入高尔基体才能被硫酸盐化，这是众所周知的 硫蛋白是一种分泌型蛋白或膜蛋白。质谱学(MS)是全球研究的有力工具 细胞和组织中的PTM分析；然而，大规模的酪氨酸O-硫化分析还不可行， 部分是由于其在质谱仪的气相环境中的不稳定性质，部分是由于缺乏 适当的数据分析策略。在MS实验中，蛋白质通常被消化成更小的多肽， 它们被电离、检测和分段以推断序列信息。当测量蛋白质时 磷酸化，另一种已知的调节细胞高尔基体拆解和重组的相当不稳定的PTM 分裂，在间期和有丝分裂高尔基体中，我们发现酪氨酸O-硫酸盐是共富集的。这一发现是 这并不奇怪，因为硫酸盐化(O-SO3)的化学性质类似于磷酸化(O-PO3H)。 然而，要推导出0.0095 Da的微小质量差，需要高质量精度的测量 在这两个PTM之间。即使这样的高性能测量正在变得更加例行公事、标准 数据库搜索工具通常不能识别蛋白质硫酸盐化，因为这种PTM在 分析。我们发现开放数据库搜索能够克服这个问题，因此，我们能够 目的：完成对大鼠肝脏高尔基体中一些新的硫蛋白的鉴定。虽然这是一个令人兴奋的进步，但 O-硫酸在蛋白水解肽中的确切位置不能直接测量。在本提案的目标1中， 我们寻求开发改进的方法来用MS检测完整的磺肽，包括消除 竞争磷酸化，多肽序列效应的测定，稳定加合物的实施， 以及选择性解离磺酸肽的条件。为了进一步允许硫酸盐站点本地化，在目标2中，我们 寻求开发在碎片离子中保留硫酸盐的同时裂解磺肽的技术。这些 方法包括负离子模式自由基启动的多肽测序，这允许磺基多肽 作为更稳定的阴离子进入质谱计，并制定“智能”数据采集策略 以改善电子转移解离。最终目标3寻求将这些改进的方法应用于 细胞和动物组织中高尔基体硫化蛋白质组的综合分析，特别是在受干扰的高尔基体下 条件，这预计会改变硫酸盐化。这些类型的测量将提供变革性的 关于酪氨酸硫化的调节作用及其对细胞功能的影响的信息。

Project Summary Tyrosine O-sulfation, i.e., transfer of a sulfonate group to tyrosine amino acid residues in proteins, is a widespread posttranslational modification (PTM) in eukaryotic cells with a variety of known functions in health and disease, including receptor binding, viral replication, inflammation, and retinal function. The enzymes that catalyze tyrosine sulfation are located in the Golgi apparatus. The function of this organelle is to ensure that correct protein modification occurs and to package proteins into vesicles for export to the cell surface, or the extracellular environment. Because proteins must typically enter the Golgi to become sulfated, most known sulfoproteins are secreted proteins or membrane proteins. Mass spectrometry (MS) is a powerful tool for global PTM analysis in cells and tissues; however, large scale analysis of tyrosine O-sulfation has not been feasible, due in part to its labile nature in the gas-phase environment of a mass spectrometer, and in part due to the lack of appropriate data analysis strategies. In MS experiments, proteins are typically digested into smaller peptides, which are ionized, detected, and fragmented to deduce sequence information. When measuring protein phosphorylation, another rather labile PTM known to regulate Golgi disassembly and reassembly during cell division, in interphase vs. mitotic Golgi, we found that tyrosine O-sulfation was co-enriched. This discovery is not surprising because the chemical properties of sulfation (O-SO3) are similar to phosphorylation (O-PO3H). However; high mass accuracy measurements are required to deduce the small mass difference of 0.0095 Da between these two PTMs. Even as such high performance measurements are becoming more routine, standard database search tools typically do not identify protein sulfation because this PTM is completely lost during analysis. We found that open database searching was able to overcome this problem and, thus, we were able to accomplish identification of a number of novel sulfoproteins in rat liver Golgi. While an exciting advance, the exact location of O-sulfation within proteolytic peptides could not be directly measured. In Aim 1 of this proposal, we seek to develop improved methods for detection of intact sulfopeptides by MS, including elimination of competing phosphorylation, determination of peptide sequence effects, implementation of stabilizing adducts, and conditions that selectively dissociate sulfopeptides. To further allow sulfate site localization, in Aim 2, we seek to develop technologies for fragmenting sulfopeptides while retaining sulfate in fragment ions. These approaches include negative ion mode free radical initiated peptide sequencing, which allows sulfopeptides to enter the mass spectrometer as more stable anions, and the development of “smart” data acquisition strategies for improved electron transfer dissociation. The final Aim 3 seeks to apply these improved approaches for comprehensive analysis of the Golgi sulfoproteome in cells and animal tissue, particularly under perturbed Golgi conditions, which are expected to alter sulfation. These types of measurements will provide transformative information regarding the regulatory roles of tyrosine sulfation and its impact on cellular function.