Enabling Mass Spectrometry Analysis of the Sulfoproteome

实现磺化蛋白质组的质谱分析

• 批准号: 10543467
• 负责人: KRISTINA HAKANSSON
• 金额: $ 29.92万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543467
• 关键词: Acidity; Alkanesulfonates; Amino Acids; Anions; Arginine; Basic Amino Acids; Cations; Cell Culture Techniques; Cell division; Cell physiology; Cell surface; Cells; Charge; Chlorates; Collaborations; Complication; Data; Data Analyses; Data Set; Databases; Detection; Development; Disease; Dissociation; Electron Transport; Electrons; Electrospray Ionization; Environment; Enzymes; Eukaryotic Cell; Free Radicals; GORASP2 gene; Gases; Golgi Apparatus; HIV; Health; Heparin; Infection; Inflammation; Interphase; Ions; Knock-out; Laboratories; Liver; Location; Lysine; Manuals; Mass Spectrum Analysis; Measurement; Measures; Membrane Proteins; Methods; Mitotic; Modality; Nature; Normal Cell; Organelles; Peptide Sequence Determination; Peptides; Performance; Phase; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Positioning Attribute; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Protein Region; Protein Secretion; Proteins; Rattus; Regulation; Reporting; Retina; Role; Site; Sulfate; Suramin; Techniques; Technology; Tissues; Trifluoroethanol; Tyrosine; Validation; Vesicle; Viral; Virus Diseases; Virus Replication; Work; absorption; adduct; animal tissue; chemical property; data acquisition; detection method; experimental study; extracellular; functional group; glycosylation; hepatoma cell; improved; inhibitor; instrument; interest; ionization; knock-down; liquid chromatography mass spectrometry; mass spectrometer; multi-photon; novel; novel strategies; peptide hormone; phosphoproteomics; prevent; protein protein interaction; protein-tyrosine sulfotransferase; receptor binding; rottlerin; sulfotransferase; sulfurtransferase; tandem mass spectrometry; tool; tyrosine O-sulfate

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.

项目摘要 酪氨酸O-硫酸化，即，将磺酸盐基团转移到蛋白质中的酪氨酸氨基酸残基，是一种 真核细胞中广泛的翻译后修饰（PTM），在健康中具有多种已知功能 和疾病，包括受体结合、病毒复制、炎症和视网膜功能。的酶 催化酪氨酸硫酸化位于高尔基体。这个细胞器的功能是确保 发生正确的蛋白质修饰并将蛋白质包装到囊泡中以输出到细胞表面，或者 细胞外环境因为蛋白质必须进入高尔基体才能被硫酸化， 硫蛋白是分泌蛋白或膜蛋白。质谱分析（MS）是全球范围内的一种强有力的工具， 细胞和组织中的PTM分析;然而，酪氨酸O-硫酸化的大规模分析尚不可行， 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较小的肽， 其被电离、检测和片段化以推断序列信息。测量蛋白质时 磷酸化，另一个相当不稳定的PTM已知调节高尔基体拆卸和重组过程中细胞 分裂，在间期与有丝分裂高尔基体中，我们发现酪氨酸O-硫酸化共富集。这一发现是 这并不奇怪，因为硫酸化（O-SO 3）的化学性质与磷酸化（O-PO 3 H）相似。 然而，需要高质量精度测量来推断0.0095 Da的小质量差 在这两个PTM之间。即使这样的高性能测量变得越来越常规，标准 数据库搜索工具通常不能识别蛋白质硫酸化，因为该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 完成大鼠肝高尔基体中一些新的磺蛋白的鉴定。虽然这是一个令人兴奋的进步， 不能直接测量O-硫酸化在蛋白水解肽内的精确位置。在本提案的目标1中， 我们寻求开发通过MS检测完整磺肽的改进方法，包括消除 竞争磷酸化，肽序列效应的测定，稳定加合物的实施， 和选择性解离磺肽的条件。为了进一步允许硫酸盐位点定位，在目标2中，我们 寻求开发用于片段化磺肽同时将硫酸根保留在片段离子中的技术。这些 方法包括负离子模式自由基引发的肽测序，其允许磺肽， 作为更稳定的阴离子进入质谱仪，并开发“智能”数据采集策略 用于改善电子转移解离。最终目标3旨在将这些改进的方法应用于 全面分析细胞和动物组织中的高尔基体磺基蛋白质组，特别是在扰动高尔基体的情况下 条件，预计将改变硫酸化。这些类型的测量将提供变革性 关于酪氨酸硫酸化的调节作用及其对细胞功能的影响。