Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.