使用液相色谱-质谱联用技术对DNA甲基化及其氧化衍生物进行定量分析。

Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS.

作者信息

Traube Franziska R, Schiffers Sarah, Carell Thomas

机构信息

Department of Chemistry, Ludwig-Maximilians-Universität München and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany.

S.S. National Institutes of Health, Bethesda, MD, USA.

出版信息

Methods Mol Biol. 2021;2272:77-94. doi: 10.1007/978-1-0716-1294-1_6.

DOI:10.1007/978-1-0716-1294-1_6

PMID:34009610

Abstract

The amount of 5-methyl-2'-deoxycytidine (mdC) and its oxidized derivatives 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify mdC, hmdC, fdC, and cadC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).

摘要

哺乳动物细胞内5-甲基-2'-脱氧胞苷（mdC）及其氧化衍生物5-羟甲基-dC（hmdC）、5-甲酰基-dC（fdC）和5-羧基-dC（cadC）的含量提供了有关细胞状态和命运的有价值信息。液相色谱-质谱（LC-MS）方法能够在低飞摩尔范围内对这些非经典DNA修饰进行可靠定量。在此，我们描述了一种广泛适用的方案，用于使用超高效液相色谱三重四极杆质谱（UHPLC-QQQ-MS）对脊椎动物来源细胞中的mdC、hmdC、fdC和cadC进行定量。