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.

项目总结