title: Chromatographic efficiency and selectivity in top-down proteomics of histones.
abstract: Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5μm in diameter, 2) superficially porous particles of 3μm in diameter with a shell of 0.2μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.
authors.affiliation: Department of Chemistry, Purdue University, United States.
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Purdue University, United States.
authors.forename: Ximo
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Northwestern University, United States.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.affiliation: Department of Chemistry, Northwestern University, United States.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Compton
authors.affiliation: Department of Chemistry, Northwestern University, United States.
authors.forename: Neil
authors.initials: N
authors.lastname: Kelleher
authors.affiliation: Department of Chemistry, Purdue University, United States. Electronic address: mwirth@purdue.edu.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Wirth
mesh_terms: D000818:Animals; D002417:Cattle; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006657:Histones / Q000032:analysis* / Q000737:chemistry*; D013058:Mass Spectrometry; D016062:Porosity; D040901:Proteomics / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D006657:Histones
keywords: Difluoroacetic acid; Histone; Post-translational modification; Revesred-phase