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个非常重要的细胞过程，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起着重要作用，尽管我们对这些作用的确切性质的理解相对较差。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，将蛋白质简化为肽集合的鸟枪蛋白质组学工作流程往往不能检测脂化肽，这可能是因为所采用的方案被优化为最容易回收的可溶性肽（蛋白质型）。解决蛋白质组学中蛋白质脂化检测的偏倚需要在分离技术方面进行新的创新，用于自下而上的方法和专门的自上而下或中间向下的方法，以完整的脂化蛋白为目标。具体目标之一将集中在脂化蛋白质的有效消化和随后的回收脂化肽通过富集与新的HILIC色谱方案。我们还将创新用于回收长链酰化肽和串联质谱的改良反相方案。总体目标将是一个强大的二维分离技术的蛋白质组范围内的检测和表征的脂化肽。具体目标二将侧重于开发自上而下的质谱技术，用于表征脂化蛋白质。这一目标包括MSMSMS策略以及中下策略，以靶向脂化位点的稳健定位。总体目标将是一个强大的质谱/蛋白质化学技术，用于自上而下分析整个蛋白质组的蛋白质脂化。 公共卫生相关性：人类基因组中编码的蛋白质的多样性执行了允许人类茁壮成长的大多数细胞功能。一些蛋白质被修饰以使它们油腻（脂肪酰化或脂化），从而期望地调节它们的功能，或者在包括一些癌症和可能的阿尔茨海默病的疾病中不期望地调节它们的功能。我们建议开发新的蛋白质组学技术，以准确地描述和测量这一重要的蛋白质修饰，迄今为止已经很难检测到。

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.