Multicolumn separation platform for simultaneous depletion and prefractionation prior to 2-DE for facilitating...

In this report, we describe an integrated fluidic platform composed of tandem affinity columns for the depletion of high-abundance proteins from human serum and on-line fractionation/concentration of medium- and low-abundance proteins by tandem immobilized metal-ion affinity chromatography (IMAC) columns and reversed phase (RP) column for in-depth proteomics analysis. The depletion columns were based on monolithic polymethacrylate with surface immobilized protein A, protein G', and antibodies for depleting the top 8 high-abundance proteins. The IMAC fractionation/concentration columns consisted of monolithic stationary phases with surface bound iminodiacetic acid (IDA) chelated with Zn2+, Ni2+ and Cu2+, while the RP column was packed with nonpolar polymer beads. The integrated multicolumn fluidic platform was very effective in reducing simultaneously both the dynamic range differences among the protein constituents of serum and the complexity of the proteomics samples, thus, facilitating the in-depth proteomics analysis by 2-DE followed by MALDI-TOF and LC-MS/MS. In fact, the number of detected spots was approximately 1450 using SYPRO fluorescent stain from which 384 spots were subsequently detected by Coomassie Blue. Since the investigation was simply a proof of concept, 295 proteins were readily identified in some selected spots by MALDI-TOF and LC-MS/MS.