Comparative Analysis of Serum Samples with the Agilent mRP-C18 Column - A New Technology for Protein Fractionation Application

Proteomic samples such as human serum are an extreme challenge to study due to the large dynamic range of the proteins of interest. The proteins in human serum can range from those considered “high-abundance” proteins, present at 2%–50% of the total protein mass (typically the μg/mL–mg/mL range) to those considered tissue biomarkers present in the pg/mL–ng/mL range. Multidimensional separation techniques have provided one way to address the complexity of these samples. A reversedphase (RP) HPLC method for the separation and fractionation of immunodepleted protein samples was developed using a novel macroporous reversed-phase (mRP) column. The optimized method and special column material combine to provide high-protein recoveries, reproducible fractionation, and higher column loads. Samples of human serum, depleted of the six most abundant proteins, were separated on a novel mRP-C18 column resulting in high protein recovery, good protein resolution, and excellent reproducibility. This allowed for a direct comparison of several immunodepleted human sera samples, which showed differences in chromatographic UV Comparative Analysis of Serum Samples with the Agilent mRP-C18 Column A New Technology for Protein Fractionation