Micro‐high‐performance liquid chromatography platform for the depletion of high‐abundance proteins and subsequent on‐line concentration/capturing of medium and low‐abundance proteins from serum. Application to profiling of protein expression in healthy and osteoarthritis sera by 2‐D gel electrophoresis

Abstract

In this investigation, an integrated microcolumn-based fluidic platform for the simultaneous depletion of high-abundance proteins and the subsequent on-line concentration/capturing of medium- and low-abundance proteins from human serum has been introduced. The platform consists of on-line coupling of tandem affinity micorcolumns to an RP microcolumn to achieve first the depletion of high-abundance proteins by the tandem affinity microcolumns followed by the concentration and capturing of medium- and low-abundance proteins by the RP microcolumn. The tandem affinity microcolumns are based on macroporous monoliths characterized by their relatively high permeability in pressure-driven flow while the RP microcolumn is packed with polymeric particles with an average particle diameter of 20 microm giving rise to a very little back pressure, thus allowing fast flow velocity across the coupled columns format and consequently short processing time of serum samples prior to analysis by 2-DE. The microcolumn-based fluidic platform was applied to serum samples from osteoarthritis (OA) donors before and after soy protein (SP) supplementation, and from healthy donors, and the resulting depleted serum samples from high-abundance proteins were profiled for protein expression by 2-DE. In general, the protein expression was lower in serum of the same OA patient after soy treatment than before soy treatment. Several proteins were down-regulated after soy treatment with transthyretin being the most affected by the SP supplementation. In addition, with respect to serum from healthy donors, the sera from OA patients showed difference in proteins expression.

Applications of proteomics in cartilage biology and osteoarthritis research

“…Analysis of serum samples from OA patients in a large populationbased study has identified an autoantibody to triosephosphate as a novel marker that could be used as a diagnostic biomarker for OA (139). A comparison of 2D protein profiles of serum from healthy individuals, OA patients and OA patients taking soy supplements (a source of anti-inflammatory compounds such as genistein) has been completed to assess the differential proteins (140). The OA serum provided evidence of altered levels of proteins including vitamin D-binding protein precursor and apolipoprotein A-I and A-IV precursors.…”

Automated Sample Preparation Platform for Mass Spectrometry-Based Plasma Proteomics and Biomarker Discovery

“…Jmeian et al , have described a novel LC system enabling capture of proteins from the depletion via a reversed phase material optimized for injections of circa 25 µL plasma [18]. In a subsequent paper he further refined the methodology, employing tandem affinity columns for the depletion and fractionation of low-abundance plasma proteins by tandem immobilized metal-ion affinity chromatography columns and capture on reversed phase (RP) column [19].…”