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.

项目摘要/摘要 在这项提案中，我们解决了现有技术在以下方面的能力方面的一个非常实质性的限制 提供来自正常组织和癌症组织的关键蛋白质组信息。在标准蛋白质组中 分析，复杂蛋白质混合物中的蛋白质，如总细胞裂解液，用 蛋白水解酶，如胰酶，以产生多肽。然后用液体分析这些多肽。 色质联用(LC-MS)，存在的蛋白质通过 其中含有多肽的存在。可以获得相对或绝对量化 如果需要，可采用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，可以很好地识别存在于复合体中的大量蛋白质。 样本，则会丢失有关特定形式的 多肽的来源是蛋白质，即“蛋白质形式”。为了更好地理解 在癌症中可操作的过程和途径，重要的是知道身份和 这些“蛋白质形式”大量存在于组织中。 最近，人们开发了一种新的方法来鉴定和定量 复杂混合物中的蛋白质形式。在这种方法中，获得了以下两条信息 样品中的每个蛋白质形式：对蛋白质形式完整质量的高精度测量， 以及它所含的赖氨酸残基的数量。该信息允许识别和 数以千计的蛋白质形式的量化。但是，目前它只能在 在含有同位素标记的赖氨酸氨基酸的培养中生长的细胞。这一限制使它成为 不可能使用该策略来鉴定癌症组织样本中的蛋白质形式。它是 建议在这里开发一种替代方法来引入所需的同位素标签 蛋白质形式定量和靶向氨基酸数量的测定 每种蛋白质形式中的残留物。已选择半胱氨酸取代赖氨酸氨基酸 酸，因为它们易于进行高效的标记反应，并且适合于同位素 标签已经设计好，并将进行合成和测试。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。 这种蛋白质形式水平的癌症变化知识将揭示一个新的宇宙 可能的癌症生物标记物，以及打开了一条目前关闭的理解 发生在癌症中的蛋白质组学变化。