title: Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry.
abstract: New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 μm I.D.) and nano (100-75 μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.
authors.affiliation: sigma-tau SpA, R&D Department, Pomezia, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Badaloni
authors.forename: Marcella
authors.initials: M
authors.lastname: Barbarino
authors.forename: Walter
authors.initials: W
authors.lastname: Cabri
authors.forename: Ilaria
authors.initials: I
authors.lastname: D'Acquarica
authors.forename: Michela
authors.initials: M
authors.lastname: Forte
authors.forename: Francesco
authors.initials: F
authors.lastname: Gasparrini
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Giorgi
authors.forename: Marco
authors.initials: M
authors.lastname: Pierini
authors.forename: Patrizia
authors.initials: P
authors.lastname: Simone
authors.forename: Ornella
authors.initials: O
authors.lastname: Ursini
authors.forename: Claudio
authors.initials: C
authors.lastname: Villani
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D016000:Cluster Analysis; D005720:Gamma Rays; D045325:HCT116 Cells; D056572:Histone Deacetylase Inhibitors / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000737:chemistry; D006801:Humans; D006877:Hydroxamic Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008689:Methacrylates / Q000737:chemistry; D058105:Polymerization / Q000528:radiation effects; D013696:Temperature; D000077337:Vorinostat
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D008689:Methacrylates; D000077337:Vorinostat