Separation of intact proteins on γ-ray-induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high-performance liquid chromatography with high-resolution mass spectrometry.

Abstract

Polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion-pair reversed-phase capillary liquid chromatography with high-resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow-split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high-resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ-ray-induced monolithic column lies in the batch-to-batch reproducibility and long-term high-temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene-divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.