Proteomic Approaches to Protein Fatty Acylation

描述(由申请人提供)：蛋白质脂肪酰化的蛋白质组学方法蛋白质脂化是一种基本的重要细胞过程，在蛋白质折叠、蛋白质靶向、膜相互作用和蛋白质/蛋白质相互作用中发挥作用。它在癌症和神经退行性疾病中发挥着重要作用，尽管我们对这些作用的确切性质的了解相对较差。新的蛋白质脂化靶点的发现可能会影响其他疾病。不幸的是，将蛋白质简化为一组多肽的鸟枪式蛋白质组学工作流程往往不能检测到脂化肽，这可能是因为所使用的方案针对最容易回收的可溶性多肽(蛋白质型)进行了优化。要解决蛋白质组学中对蛋白质脂化检测的偏见，需要在自下而上方法的分离技术和针对完整脂化蛋白质的专门的自上而下或中间向下的分离技术方面进行创新。具体目标一将重点放在脂化蛋白质的有效消化和随后通过新的HILIC层析程序富集回收脂化多肽。我们还将创新改进的反相方法，用于回收长链酰化多肽和串联质谱学。总体目标将是一种强大的二维分离技术，用于蛋白质组范围的脂化多肽的检测和表征。具体目标二将重点开发用于表征脂化蛋白质的自上而下的质谱学技术。这一目标包括MSMSMS策略以及针对脂化位点的稳健本地化的中下策略。总体目标将是一个强大的质谱学/蛋白质化学技术，用于对蛋白质组中的蛋白质脂肪作用进行自上而下的分析。 与公共卫生相关：人类基因组中编码的蛋白质的多样性执行了使人类茁壮成长的大部分细胞功能。一些蛋白质被修饰以使它们变得油腻(脂肪酰化或脂化)，从而调节它们的功能，或者在包括一些癌症和可能不希望的阿尔茨海默氏症在内的疾病中调节其功能。我们建议开发新的蛋白质组学技术来准确描述和测量到目前为止检测不到的这一重要的蛋白质修饰。

DESCRIPTION (provided by applicant): Proteomic approaches to protein fatty acylation Protein lipidation is a fundamentally important cellular process that functions in protein folding, protein targeting, membrane interaction and protein/protein interactions. It plays significant roles in cancer and neurodegenerative disease though our understanding of the exact nature of these roles is relatively poor. Discovery of new protein lipidation targets will potentially impact other diseases. Unfortunately, shotgun proteomics workflows that simplify proteins to a collection of peptides tend not to detect lipidated peptides, probably because the protocols employed are optimized to the most easily recovered soluble peptides (proteotypic). Addressing the bias against protein lipidation detection in proteomics requires novel innovations in separations technologies for bottom-up approaches and specialized top-down or middle-down approaches when targeting intact lipidated proteins. Specific aim one will focus on efficient digestion of lipidated proteins and subsequent recovery of lipidated peptides via enrichment with novel HILIC chromatography protocols. We will also innovate modified reverse-phase protocols for recovery of long- chain acylated peptides and tandem mass spectrometry. The overall goal will be a robust two-dimensional separation technology for proteome-wide detection and characterization of lipidated peptides. Specific aim two will focus development of top-down mass spectrometry technologies for characterization of lipidated proteins. This aim includes MSMSMS strategies as well as middle-down strategies to target robust localization of lipidation sites. The overall goal will be a robust mass spectrometry /protein chemistry technology for top-down analysis of protein lipidation across the proteome. PUBLIC HEALTH RELEVANCE: The diversity of proteins coded in the human genome perform the majority of cellular functions that allow human beings to thrive. Some proteins are modified to make them greasy (fatty acylation or lipidation) thereby modulating their function desirably, or in diseases including some cancers and possibly Alzheimer's Disease undesirably. We propose to develop new proteomics technologies to accurately describe and measure this important protein modification that has to date been poorly detected.