MASS SPECTROMETRY OF ACYLATED PEPTIDES

酰化肽的质谱分析

• 批准号: 7601939
• 负责人: Catherine E. Costello
• 金额: $ 0.43万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601939
• 关键词: Acylation; Anhydrides; Biological; Bos taurus; Cattle; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Digestion; Electrospray Ionization; Eledoisin; Esters; Funding; GTP-Binding Proteins; Glutathione; Glycine; Grant; Institution; Ions; Length; Lysine; Mass Spectrum Analysis; Membrane; Myelin P0 Protein; N-terminal; Pathogenesis; Pattern; Peptides; Proteins; Reaction; Research; Research Personnel; Resources; Sampling; Screening procedure; Serine; Signal Transduction; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substance P; Techniques; Tissues; Ubiquitin; United States National Institutes of Health; Work; acyl group; carbonate dehydratase; ghrelin; palmitoyl chloride; tandem mass spectrometry; thioester

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acylation of proteins and peptides is associated with signal transduction, membrane function and bacterial pathogenesis. Fatty acyl groups of varying lengths (eg. octanoyl, myristoyl, palmitoyl and stearoyl) have been found to co- and post-translationally modify proteins and peptides such as G-proteins and ghrelin. Acylation sites include N-terminal glycine and lysine, cysteine and serine residues via amino, thioester and ester linkages respectively. We have examined the fragmentation patterns for naturally occurring and synthetic acylated peptides and proteins using ESI and MALDI tandem mass spectrometry with the aim of developing screening techniques for the analysis of biological samples. Peptides were synthetically prepared by reaction with octanoyl, myristoyl or palmitoyl chloride. Ubiquitin and bovine carbonic anhydrase were reacted with anhydrides or N-hydroxysuccinimidyl esters. Myelin protein P0, Ghrelin and the synthetically acylated peptides including substance P, glutathione and eledoisin were analyzed by MALDI-TOF, MALDI-Q-o-TOF and ESI-Q-o-TOF mass spectrometry. It was found that acylated peptides required slightly higher collisional energies when compared with the corresponding unacylated peptide in the ESI-MS/MS analysis. It was also found that higher collision energies were required with increasing chain length of the acyl moiety. Characteristic ions, corresponding to acylated immonium ions were readily identified in MALDI and ESI tandem mass spectra. MALDI-PSD in contrast did not always yield characteristic immonium ions. These characteristic immonioum ions were used to confirm the sites of acylation in synthetically acylated ubiquitin and bovine carbonic anhydrase following enzymatic digestion and LC-MS/MS analysis. This work will be extended to the examination of biological tissues for the presence of acylated peptides and proteins.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 蛋白质和肽的酰化与信号转导、膜功能和细菌发病机制有关。不同长度的脂肪酰基（例如辛酰基、肉豆蔻酰基、棕榈酰基和硬脂酰基）被发现可以对蛋白质和肽（例如 G 蛋白和生长素释放肽）进行共翻译和翻译后修饰。酰化位点包括分别通过氨基、硫酯和酯键连接的N-末端甘氨酸和赖氨酸、半胱氨酸和丝氨酸残基。我们使用 ESI 和 MALDI 串联质谱法检查了天然存在和合成的酰化肽和蛋白质的断裂模式，旨在开发用于分析生物样品的筛选技术。 通过与辛酰氯、肉豆蔻酰氯或棕榈酰氯反应合成制备肽。泛素和牛碳酸酐酶与酸酐或N-羟基琥珀酰亚胺酯反应。 采用 MALDI-TOF、MALDI-Q-o-TOF 和 ESI-Q-o-TOF 质谱分析髓磷脂蛋白 P0、Ghrelin 以及合成的酰化肽（包括 P 物质、谷胱甘肽和香皂素）。在 ESI-MS/MS 分析中发现，与相应的未酰化肽相比，酰化肽需要稍高的碰撞能量。还发现，随着酰基部分链长的增加，需要更高的碰撞能量。在 MALDI 和 ESI 串联质谱中很容易识别出对应于酰化铵离子的特征离子。相比之下，MALDI-PSD 并不总是产生特征性的铵离子。在酶消化和 LC-MS/MS 分析后，使用这些特征性亚铵离子来确认合成酰化泛素和牛碳酸酐酶中的酰化位点。这项工作将扩展到检查生物组织中是否存在酰化肽和蛋白质。