Enabling Mass Spectrometry Analysis of the Sulfoproteome

实现磺化蛋白质组的质谱分析

基本信息

批准号：
10096628
负责人：
KRISTINA HAKANSSON
金额：
$ 30.01万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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/10096628
关键词：
Acidity Alkanesulfonates Amino Acids Anions 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 Ensure Environment Enzymes Eukaryotic Cell Free Radicals GORASP2 gene Gases Golgi Apparatus Health Heparin Infection Inflammation Interphase Ions Knock-out Laboratories Liver Location Manuals Mass Spectrum Analysis Measurement Measures Membrane Proteins Methods Mitotic Nature Normal Cell Organelles Peptide Sequence Determination Peptides Performance Phase Phosphopeptides Phosphoric Monoester Hydrolases Phosphorylation Play Positioning Attribute Post-Translational Protein Processing Primary carcinoma of the liver cells Process 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 arginyllysine base chemical property data acquisition detection method experimental study extracellular functional group glycosylation improved inhibitor/antagonist instrument interest ionization knock-down liquid chromatography mass spectrometry mass spectrometer novel novel strategies peptide hormone phosphoproteomics prevent protein protein interaction protein-tyrosine sulfotransferase receptor binding rottlerin searchable database sulfotransferase sulfurtransferase tandem mass spectrometry tool tyrosine O-sulfate

项目摘要

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.

项目概要酪氨酸O-硫酸化，即将磺酸基转移到蛋白质中的酪氨酸氨基酸残基上，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和疾病，包括受体结合、病毒复制、炎症和视网膜功能。催化酪氨酸硫酸化位于高尔基体中，该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，或由于蛋白质通常必须进入高尔基体才能被硫酸化，因此最为人所知。硫蛋白是分泌蛋白或膜蛋白，质谱 (MS) 是全球的强大工具。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气相环境中的不稳定性质，部分是由于缺乏在 MS 实验中，蛋白质通常被消化成更小的肽，在测量蛋白质时，它们被电离、检测和片段化以推断序列信息。磷酸化，另一种相当不稳定的 PTM，已知可调节细胞过程中高尔基体的分解和重组在分裂间期与有丝分裂高尔基体中，我们发现酪氨酸O-硫酸化是共同富集的。这一发现是。这并不奇怪，因为硫酸化 (O-SO3) 的化学性质与磷酸化 (O-PO3H) 相似。然而，需要高质量精度测量才能推导出 0.0095 Da 的小质量差。尽管这种高性能测量变得更加常规、标准。数据库搜索工具通常无法识别蛋白质硫酸化，因为该 PTM 在检测过程中完全丢失。我们发现开放数据库搜索能够克服这个问题，因此我们能够做到这一点。完成大鼠肝脏高尔基体中多种新型硫蛋白的鉴定，这是一项令人兴奋的进展。在该提案的目标 1 中，无法直接测量蛋白水解肽中 O-硫酸化的确切位置。我们寻求开发通过 MS 检测完整磺肽的改进方法，包括消除竞争性磷酸化，肽序列效应的测定，稳定加合物的实施，以及选择性解离硫肽的条件为了进一步实现硫酸盐位点定位，在目标 2 中，我们寻求开发碎裂硫肽同时将硫酸盐保留在碎片离子中的技术。方法包括负离子模式自由基引发的肽测序，它允许磺肽作为更稳定的阴离子进入质谱仪，以及“智能”数据采集策略的开发最终目标 3 旨在应用这些改进的方法。细胞和动物组织中高尔基磺基蛋白质组的综合分析，特别是在高尔基受到干扰的情况下预计会改变硫酸盐化的条件，这些类型的测量将提供变革性的结果。有关酪氨酸硫酸化的调节作用及其对细胞功能影响的信息。