Capillary electrophoresis of histone H1 variants at neutral pH in dynamically modified fused- silica tubing

Abstract

Existing methods for the analysis of histone H1 by capillary electrophoresis (CE) employ acidic buffers (pH <3.0) to suppress silanol ionization and minimize the loss of these extremely basic proteins by adsorption to capillary walls. Here we describe the use of Polybrene (PB) as a dynamic modification reagent in a simple procedure that facilitates the analysis of chicken H1 at neutral pH. PB is adsorbed to the inner surfaces of capillaries to render them cationic prior to use and a low concentration of PB is included in the electrolyte to replenish the coating during use. Inclusion of ethylenediaminetetraacetic acid (EDTA) in the electrolyte results in the assembly of a dynamic cation-exchange layer upon the immobilized PB that influences the relative mobilities of H1 variants. The six nonallelic variants of H1 known in this species as well as certain allelic variants are resolved. Because the procedure is effective in preventing the adsorption of proteins as basic as H1 at neutral pH, this strategy should facilitate CE analyses of many basic proteins under conditions that maintain their native conformation.

The significance, development and progress of high-throughput combinatorial histone code analysis

“…Wiktorowicz [53] used a charge reversal strategy to repel histones from the capillary surface to achieve separation of differentially acetylated forms of H4 for the first time by HPCE. The work of Mizzen [54] introduces some degree of cation exchange behavior to the capillary surface. Related concepts arise later in liquid chromatography separations.…”

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

“…Gurley, Lindner and Mizzen separately demonstrated HPCE methods to efficiently separate histones [101][102][103][104][105][106][107].…”

Capillary electrophoresis of proteins 1999-2001

“…It is less stable at low ionic strength (50.003), in the presence of SDS and after an application of a hydrophobic analyte such as polystyrene carbonate [40]. EOF and adsorption of proteins to the capillary wall can be manipulated by other dynamic wall coatings as published during the last two years: fluorosurfactants [42,43], polyvinyl alcohol (PVA) [44], tentacle-like oligourethanes [45], putrescine [46], Pluronic liquid crystals [47,48], sulfonated polymers [49], Polybrene [50], linear hydrocarbons and ethylbenzene [51], glycerylpropyltrimethoxysilane [52].…”

“…These applications include analysis of chemokine stromal cell-derived factor-1 [181], soluble chromatin from chicken erythrocytes [182], jellyfish venom proteins [183], sialoglycoproteins [184], turkey egg ovalbumin glycoforms [57], cathepsin D [185], bovine collagens [116], fluorescent adduct formed in vivo in collagen [71], caseinoglycomacropeptide in cosmetic lotions [186], polyethylene glycol-modified proteins (including hemoglobin as a potential blood substitute) [187], salmon calcitonin [188], ribonuclease and lysozyme [189], further transmembrane serine receptor [190], salivary a-amylase [191], outer membrane proteins from Proteus penneri [192], granulocyte colonystimulating factor [193], insulin antibodies [194], staphylococcal enterotoxin A [195], green fluorescent proteins in human embryonic kidney cells [196], peroxidase:antiperoxidase immune complexes [197], a(2u)-globulin in rat kidney cytosol [198], histone H1 variants [50], dilute proteins in formulations containing interfering excipients [199], subviral particles of human rhinovirus serotype 2 [200], seed 2S albumins [201], mutant a-and b-globulin chains [202], abnormal prion proteins [203] proteins interacting with DNA [204], alkaline phosphatase inhibited by theophylline [205], proteins in single cells [70], determination of Src kinase and kinase C activity [206], and bilirubin oxidase activity [207].…”