Abstract
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.
Original language | English (US) |
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Article number | e0240829 |
Journal | PloS one |
Volume | 15 |
Issue number | 10 October |
DOIs | |
State | Published - Oct 2020 |
Funding
This work was carried out with a financial support from The Paul G. Allen Family Foundation (Award # 11715), NCI CCSG P30CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, and the National Resource for Translational and Developmental Proteomics supported by P41GM108569 to NLK. We would also like to acknowledge funding from NCI K08CA169055, UVA Cancer Center through the NCI Cancer Center Support Grant P30CA44579, the University of Virginia and the American Society of Hematology (ASHAMFDP-20121) under the ASHAMFDP partnership with The Robert Wood Johnson Foundation to FEG-B. This study was conducted by the ECOG-ACRIN Cancer Research Group (Peter J. O'Dwyer, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: CA180827, CA180820, CA233290, CA189859, CA233321, CA233270. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
ASJC Scopus subject areas
- General