Departments of Chemistry, Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, Evanston, IL, United States of America.
Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, United States of America.
PLoS One. 2020 Oct 26;15(10):e0240829. doi: 10.1371/journal.pone.0240829. eCollection 2020.
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.
组蛋白翻译后修饰（PTMs）为维持细胞内染色体完整性提供了一种强大的调控机制。组蛋白乙酰化和甲基化是研究最广泛的组蛋白 PTMs，它们与染色质相关蛋白协同作用，控制转录过程中遗传信息的可及性。细胞内组蛋白 PTM 的改变与疾病状态有关，具有重要的生物标志物和治疗潜力。传统的基于自上而下的组蛋白质谱需要大量的细胞，通常需要一百万或更多。然而，对于某些细胞亚型特异性研究，很难或不可能获得如此大量的细胞，并且稀有组蛋白 PTM 的定量通常是无法实现的。本研究建立了一种靶向 LC-MS/MS 方法，用于定量分析细胞系和原代人标本中的组蛋白 PTMs 丰度。通过省略核分离和减少组蛋白衍生化轮次来改进样品制备，以提高对低至 1000 个细胞的组蛋白肽的检测。在本研究中，我们开发并验证了一种针对 75 种组蛋白肽的靶向组蛋白分析的定量 LC-MS/MS 方法，该方法只需 10000 个细胞即可进行分析。此外，我们能够从仅 1000 个人原代干细胞中检测和定量 61 种组蛋白肽。从 1000 个急性髓系白血病患者细胞中可以检测到 37 种组蛋白肽。我们预计，这种改进的方法可以用于许多难以获得大量细胞的应用中，包括稀有人类细胞群体。
