Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Abstract

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

Influence of mixed electrolytes on the adsorption of lysozyme, PEG, and PEGylated lysozyme on a hydrophobic interaction chromatography resin

“…Hydrophobic interaction chromatography (HIC) is an important method for the separation of biological macromolecules . To enhance the adsorption in HIC, salts are used.…”

“…Hydrophobic interaction chromatography (HIC) is an important method for the separation of biological macromolecules. [1][2][3] To enhance the adsorption in HIC, salts are used. Choosing suitable salts for HIC and their concentration is a tedious task, as it has to be done by experimental screening.…”

Sample displacement chromatography of plasmid DNA isoforms

“…SDC approach should optimally be performed under overloading conditions, since the main separation occurs already during sample loading and column capacity is thus optimally used [8]. SDC was introduced for preparative purification of peptides in reverse-phase mode [9] and has subsequently been successfully applied for purification of proteins in ion-exchange [10], affinity [11] and HIC [12] modes, using traditional porous particle chromatographic media, http operating under diffusion mass transfer conditions. SDC approach is most useful for samples where the product binds to a chromatographic support more strongly than the impurities, because this enables fast and efficient purification of complex samples [8,11].…”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…The enrichment of low‐abundance proteins from a complex samples such as blood plasma/serum can be achieved by sample displacement chromatography on monolithic supports without use of expensive antibodies. Application of sample displacement chromatography in HIC mode and IEC mode for enrichment/depletion of proteins from human plasma were described.…”