Development of MassSQUIRM to Quantitatively Measure Lysine Methylation

描述(申请人提供)：组蛋白赖氨酸残基的甲基化与多种癌症表型有关。组蛋白赖氨酸残基可以添加多达三个甲基，并且每种状态都可以具有明显不同的细胞角色。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，这些技术既不全面，也不定量。绕过这些限制的另一种方法是直接使用质谱学，但这种方法的问题是，差异甲基化的多肽不会电离相同，也不能作为活性测量的直接比较。因此，癌症表观遗传学研究和整个表观遗传学领域需要一种全面的方法来同时定量地监测脱甲基酶/甲基转移酶反应中间产物(即赖氨酸上的不同甲基化状态)。2011年，我们提供了一种新的方法来全面和定量地测量赖氨酸甲基化状态，称为MassSQUIRM(使用同位素还原甲基化的质谱学定量)。MassSQUIRM利用赖氨酸上同位素重甲基的化学结合，将所有反应中间产物(非甲基和单甲基)转化为全二甲基赖氨酸(仅区别于氢和氢-这不会影响质谱学中的电离性质)。通过比较重物种和轻物种混合物的多肽强度，可以对赖氨酸甲基化进行全面的(非、单甲基和二甲基态)定量。我们最近发表了MassSQUIRM技术，在这项申请中，我们概述了如何在与癌症相关的背景下对其进行评估，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一套MassSQUIRM试剂盒，以检测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(特别是非、单甲和双甲基化)。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：目的1.通过分析一组组蛋白赖氨酸去甲基酶和甲基转移酶来确定MassSQUIRM的普遍适用性。目的2.评价MassSQUIRM检测细胞裂解物中LSD1活性的有效性。目的3.用一组LSD1抑制剂对MassSQUIRM进行赖氨酸去甲基化筛选。

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.