Development of MassSQUIRM to Quantitatively Measure Lysine Methylation

描述（由申请人提供）：组蛋白赖氨酸残基的甲基化与许多癌症表型相关。组蛋白赖氨酸残基可以添加多达三个甲基，每种状态可以具有明显不同的细胞作用。多年来，表观遗传学的研究人员（包括那些专注于癌症表观遗传学过程的研究人员）一直使用依赖抗体或放射性的技术来测量给定赖氨酸的甲基化状态；然而，这些既不全面也不定量。绕过这些限制的另一种方法是直接使用质谱法，但这种方法的问题是，差异甲基化肽的电离程度不同，不能直接比较作为活性的测量。因此，癌症表观遗传学研究和整个表观遗传学领域都需要一种综合的方法来同时定量监测去甲基化酶/甲基转移酶反应中间体（即赖氨酸上的不同甲基态）。在2011年，我们提供了一种新的方法来全面和定量测量赖氨酸甲基化状态，称为MassSQUIRM（质谱定量使用同位素还原甲基化）。MassSQUIRM利用同位素重甲基在赖氨酸上的化学结合，将所有反应中间体（非甲基和单甲基）转化为全二甲基赖氨酸（仅因氢和氘而不同-不影响质谱中的电离特性）。重和轻的物种的混合物的肽强度的比较允许赖氨酸甲基化的全面（非，单和二甲基状态）定量。我们最近发布了MassSQUIRM技术，在这个应用程序中，我们概述了我们将如何在癌症相关的背景下评估它，以便最终开发一种用于癌症研究的试剂盒。我们的总体目标是为癌症研究人员提供MassSQUIRM试剂盒，以测定与特定癌症表型相关的蛋白质中赖氨酸残基的去甲基化和甲基化（非甲基化，单甲基化和二甲基化）。为了验证MassSQUIRM方法在癌症研究中的应用，我们将追求以下目标：通过测定一组组蛋白赖氨酸去甲基化酶和甲基转移酶来确定MassSQUIRM的一般适用性。目标2。评估使用MassSQUIRM检测细胞裂解物中LSD1活性的有效性。目标3。优化MassSQUIRM赖氨酸去甲基化筛选与一组LSD1抑制剂。

DESCRIPTION (provided by applicant): The methylation of histone lysine residues has been correlated to numerous phenotypes of cancer. Histone lysine residues can have up to three methyls added and each state can have clearly different cellular roles. For years, researchers in epigenetics (including those focused on epigenetic processes in cancer) have used techniques that rely on antibodies or radioactivity to measure the methylation state of a given lysine; however, these are neither comprehensive nor quantitative. What has become an alternative approach to get around these limitations is the direct use of mass spectrometry, but the problem with this approach is that differentially methylated peptides do not ionize the same and CANNOT be directly compared as a measurement of activity. Therefore, the field of cancer epigenetic research and epigenetics as a whole needed a comprehensive method to simultaneously monitor demethylase/methyltransferase reaction intermediates (i.e., different methyl states on a lysine) in a quantitative manner. In 2011, we provided a novel approach for the comprehensive and quantitative measurement of lysine methylation states, which is called MassSQUIRM (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation). MassSQUIRM utilizes the chemical incorporation of isotopically heavy methyl groups on lysines to convert all reaction intermediates (un- and monomethyl) to fully dimethyl lysines (differing only by hydrogen and deuterium - which does not affect ionization properties in mass spectrometry). A comparison of peptide intensities of the mixture of heavy and light species allows for comprehensive (un-, mono- and dimethyl states) quantitation of lysine methylation. We recently published the MassSQUIRM technique, and in this application we outline how we will evaluate it in a cancer relevant context in order to ultimately develop a kit for cancer research. Our overall goal is to provide a MassSQUIRM kit to cancer researchers to assay demethylation and methylation (un-, mono- and dimethylation specifically) of lysine residues in proteins correlated to particular cancer phenotypes. To validate the MassSQUIRM approach for its use in cancer research, we will pursue the following Aims: Aim 1. Determine the general applicability of MassSQUIRM by assaying a panel of histone lysine demethylases and methyltransferases. Aim 2. Evaluate the effectiveness of using MassSQUIRM to assay LSD1 activity from cell lysates. Aim 3. Optimize MassSQUIRM for lysine demethylation screening with a panel of LSD1 inhibitors.