Novel NeuCode Tagging Reagents for Identification and Quantification of Intact Proteoforms in Cancer Tissues

用于癌症组织中完整蛋白质形式的识别和定量的新型 NeuCode 标记试剂

• 批准号: 9443408
• 负责人: LLOYD M SMITH
• 金额: $ 22.63万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-14 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9443408
• 关键词: Address; Amino Acids; Cancer Biology; Categories; Cell Culture Techniques; Cells; Chemistry; Complex; Complex Mixtures; Cysteine; Human; Isotope Labeling; Isotopes; Knowledge; Laboratories; Light; Lysine; Malignant Neoplasms; Measurement; Molecular; Motivation; Neutrons; Normal tissue morphology; PC3 cell line; Pathway interactions; Peptide Hydrolases; Peptides; Process; Prostate; Prostatic Neoplasms; Proteins; Proteomics; Reaction; Reagent; Research; Resolution; Sampling; Screening for cancer; Specificity; Stable Isotope Labeling; Technology; Testing; Tissue Sample; Tissues; Trypsin; Variant; Work; Yeasts; base; cancer biomarkers; clinically relevant; design; liquid chromatography mass spectrometry; novel; novel strategies; professor; protein complex; success

Project Summary/Abstract In this proposal we address a very substantial limitation in the ability of existing technologies to provide critical proteomic information from normal and cancer tissues. In standard proteomic analyses, proteins in complex protein mixtures such as total cell lysate are digested with proteases such as trypsin to produce peptides. These peptides are then analyzed by liquid chromatography and mass spectrometry (LC-MS), and the proteins present are identified by the presence of peptides contained within them. Relative or absolute quantification may be obtained if desired by a variety of different isotopic tagging strategies. While this strategy, termed “bottom-up” proteomics, works well for identifying large numbers of proteins present in complex samples, it suffers from the loss of contextual information about the particular form of the proteins from which the peptides are derived, the “proteoforms”. In order to understand the processes and pathways that are operative in cancer, it is essential to know the identities and abundances of these “proteoforms” present in the tissues. Recently, a new approach has been developed for the identification and quantification of proteoforms in complex mixtures. In this approach two pieces of information are obtained for each proteoform in the sample: a highly accurate measurement of the proteoform intact mass, and the number of lysine residues it contains. This information allows identification and quantification of thousands of proteoforms. However, at present it can only be performed on cells grown in culture containing isotopically labeled lysine amino acids. This limitation makes it impossible to use the strategy for identification of proteoforms in cancer tissue samples. It is proposed here to develop an alternative means of introducing the isotopic tags needed for proteoform quantification and for the determination of the number of a targeted amino acid residue in each proteoform. Cysteine amino acids have been chosen in place of lysine amino acids because of their amenability to highly efficient tagging reactions, and suitable isotopic labels have been designed and will be synthesized and tested. This new chemistry will provide the power of the isotopic tagging strategy for proteomic analyses of cancer tissue samples. Such proteoform level knowledge of changes that occur in cancer will reveal a new universe of possible cancer biomarkers, as well as opening a currently closed avenue to understanding the proteomic changes that occur in cancer.

项目总结/摘要 在这个建议中，我们解决了现有技术能力的一个非常大的限制， 从正常和癌症组织中提供关键的蛋白质组学信息。在标准蛋白质组学中 分析中，复杂蛋白质混合物（例如总细胞裂解物）中的蛋白质被消化， 蛋白酶如胰蛋白酶以产生肽。然后通过液相色谱法分析这些肽。 通过高效液相色谱和质谱（LC-MS）分析，并通过质谱鉴定存在的蛋白质。 其中所含的肽的存在。可获得相对或绝对定量 如果需要的话，通过各种不同的同位素标记策略。虽然这一战略被称为 “自下而上”的蛋白质组学，适用于鉴定复杂蛋白质中存在的大量蛋白质。 样本，它遭受损失的上下文信息的特定形式的 肽衍生自的蛋白质，即“蛋白形式”。为了解 在癌症中起作用的过程和途径，了解其身份和 这些“蛋白形式”的丰度存在于组织中。 最近，已经开发了一种新的方法来识别和量化 复杂混合物中的蛋白形式。在这种方法中，获得两条信息， 样品中的每种蛋白质型：蛋白质型完整质量的高度精确测量， 以及它所含的赖氨酸残基的数量。这些信息有助于识别和 对数以千计的蛋白质进行定量。然而，目前它只能在 在含有同位素标记的赖氨酸氨基酸的培养物中生长的细胞。这种局限性使其 不可能使用该策略来鉴定癌组织样品中的蛋白质型。是 这里提出了一种替代方法，引入所需的同位素标签， 蛋白质型定量和用于测定目标氨基酸的数量 每种蛋白质中的残基。选择半胱氨酸氨基酸代替赖氨酸氨基酸 酸，因为它们对高效标记反应的顺从性，以及合适的同位素 标签已经设计，将进行合成和测试。这种新的化学物质将提供 同位素标记策略在癌症组织样本蛋白质组学分析中的作用。 对癌症中发生的变化的这种蛋白质形式水平的知识将揭示一个新的宇宙， 可能的癌症生物标志物，以及打开一个目前封闭的途径，以了解 癌症中发生的蛋白质组变化。