PNNL

Abstract

We report on a customized mouse specific SuperMix immunoaffinity column and strategy for separating low abundance proteins from high and moderate abundance proteins in mouse plasma. When applied in tandem with a mouse IgY7 column that removes the seven most abundant proteins in blood, the SuperMix column captures >100 additional moderate abundance proteins, thus allowing significant enrichment of low abundance proteins in the flow-through fraction. A side-by-side comparison of results obtained from 2D-LC-MS/MS analyses of flow-through samples from IgY7 and SuperMix columns revealed a nearly two-fold improvement in the overall proteome coverage. Detection of low abundance proteins was also enhanced, as evidenced by a more than two-fold increase in the coverage of cytokines, growth factors, and other low abundance proteins. Moreover, the tandem separations are automated, reproducible, and allow effective identification of protein abundance differences from LC-MS/MS analyses. Considering the overall reproducibility and increased sensitivity using the IgY7-SuperMix separation system, we anticipate broad applications of this strategy for biomarker discovery using mouse models.