Fabrication of a monolithic, macroporous diallyl maleate-based material and its application for fast separation of intact proteins from human plasma with reversed-phase chromatography.

Abstract

A monolithic diallyl maleate-based column was developed and used to separate intact proteins from complex bio-samples with high-performance liquid chromatography. The resulting monolith exhibited a relatively uniform porous structure with macro-pores. A sample of standard proteins mixture was used to explore the reversed-phase mechanism for fast separation on the homemade monolithic column. The observed retention time increased with the values of the diameters, relative hydrophobicities and molecular weights of the standard proteins, which mainly depends on the relative hydrophobicity of the proteins. It is worth mentioning that the mask of the three most abundant proteins in human plasma can be avoided, thus providing an opportunity for the middle- and low-abundance proteins to be detected. According to this exploration, the fast separation of human plasma proteins on the diallyl maleate-based monolithic column was achieved in 15\u2009min. The chromatographic fractions were identified by liquid chromatography-tandem mass spectrometry, and the results indicate that the present method is an outstanding method for the fast and efficient fractionation of human plasma that will be significant sense for plasma proteomics research, especially for exploring new disease marker and drug target.