Targeted detection and quantitation of histone modifications from 1,000 cells.
Targeted detection and quantitation of histone modifications from 1,000 cells.
复制标题
对1,000个细胞的组蛋白修饰的靶向检测和定量。
DOI：
10.1371/journal.pone.0240829
复制
发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Kelleher NL
中科院分区：
文献类型：
--
作者：
Abshiru NA;Sikora JW;Camarillo JM;Morris JA;Compton PD;Lee T;Neelamraju Y;Haddox S;Sheridan C;Carroll M;Cripe LD;Tallman MS;Paietta EM;Melnick AM;Thomas PM;Garrett-Bakelman FE;Kelleher NL
组蛋白翻译后修饰(PTM)为维持细胞中染色体的完整性创造了一种强大的调节机制。组蛋白乙酰化和甲基化是研究最广泛的组蛋白PTM，它们与染色质相关蛋白协同作用，在转录过程中控制遗传信息的获取。细胞组蛋白PTM的改变与疾病状态有关，并具有关键的生物标志物和治疗潜力。传统的自下而上的组蛋白质谱学需要大量的细胞，通常是一百万个或更多。然而，对于一些细胞亚型特异性的研究，很难或不可能获得如此大量的细胞，而且罕见的组蛋白PTM的量化往往是无法实现的。建立了一种靶向LC-MS/MS方法来定量从细胞系和原代人类标本中提取的组蛋白PTM的丰度。通过省略核分离和减少组蛋白衍生化次数来改进样品制备，以改进组蛋白多肽的检测，最低可达1000个细胞。在目前的研究中，我们开发并验证了一种定量LC-MS/MS方法，该方法专为具有多达10,000个细胞的75个组蛋白多肽的靶向组蛋白分析而定制。此外，我们能够从1000个原代人类干细胞中检测并量化61个组蛋白多肽。从1000个急性髓系白血病患者细胞中检测到37个组蛋白多肽是可能的。我们预计，这种修改后的方法可以用于许多具有挑战性的应用中，包括稀有的人类细胞群体。
Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.
登录
查看更多内容
影响因子：
9.2
作者：
Peterson, CL;Laniel, MA
通讯作者：
Laniel, MA
影响因子：
14.8
作者：
Garcia, Benjamin A.;Mollah, Sahana;Hunt, Donald F.
通讯作者：
Hunt, Donald F.
影响因子：
4
作者：
Kouzarides, T
通讯作者：
Kouzarides, T
影响因子：
7
作者：
Peach, Sally E.;Rudomin, Emily L.;Jaffe, Jacob D.
通讯作者：
Jaffe, Jacob D.
影响因子：
82.9
作者：
Piunti A;Hashizume R;Morgan MA;Bartom ET;Horbinski CM;Marshall SA;Rendleman EJ;Ma Q;Takahashi YH;Woodfin AR;Misharin AV;Abshiru NA;Lulla RR;Saratsis AM;Kelleher NL;James CD;Shilatifard A
通讯作者：
Shilatifard A