期刊
JOURNAL OF PROTEOME RESEARCH
卷 9, 期 10, 页码 5501-5509出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr100497a
关键词
histones; post-translational modifications; ultra-high performance liquid chromatography; mass spectrometry; label-free quantification
资金
- CEA
- French National Research Agency [ANR-07-B1AN-0098-01]
- French Association for Research on Cancer [ARC 9916]
Histones are subjected to extensive post-translational modifications (PTMs) that are known to play key roles in many biological processes. In this study, we report a fast, efficient, highly reproducible, and easily automated method involving ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution/high mass accuracy LTQ-Orbitrap mass spectrometer to profile core histone modifications/variants from WI-38 primary human fibroblasts. The whole analysis was performed on intact unfractionated histones within 19 min, which is similar to 3-fold faster than previously published procedures. High mass accuracy measurements combined with top-down tandem mass spectrometry (MS) experiments enable accurate histone identification. Experimental and biological variations were thoroughly assessed and were 8% and 16% on average, respectively. With a sample preparation reduced to the minimum, characterization of the most abundant histones can be achieved in a single experiment. Semi-quantitative information can be obtained with respect to the relative abundances of the detected isoforms through a label-free approach. lsoform identities and relative distributions were further confirmed by the LC-MS/MS analysis of tryptic digests. Overall, our UHPLC MS approach for histone profiling offers a sensitive and reproducible tool that will be of great value for exploring PTMs and variants and can readily be applied to clinical or pharmaceutical studies.
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