Ultra-high performance liquid chromatography-mass spectrometry for the fast profiling of histone post-translational modifications.
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 ∼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. Isoform 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.
Contrepois
Kévin
K
CEA, iBiTecS, Service de Biologie Intégrative et Génétique Moléculaire (SBIGeM), Gif Sur Yvette, France.
Ezan
Eric
E
Mann
Carl
C
Fenaille
François
F
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006657:Histones / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis; D020033:Protein Isoforms / Q000032:analysis; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006657:Histones; D010750:Phosphoproteins; D020033:Protein Isoforms