PNNL

Abstract

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomic technologies as it makes it especially difficult to detect low-abundant proteins in human biofluids such as blood plasma, an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low-abundant proteins in human plasma. The tandem IgY12-SuperMix system separates ~60 abundant proteins from those low abundant proteins in plasma, thus allowing significant enrichment of low-abundance plasma proteins in the flow-through fraction. High reproducibility of the tandem separations in both sample processing recovery and LC-MS/MS identification results were observed, comparable to the results from the single IgY12 separations. Tandem SuperMix flow-through samples based on single dimensional (1D) and two dimensional (2D) LC-MS/MS analyses revealed a 60-80% improvement in overall proteome coverage when compared to the single IgY12 flow-through, suggesting significantly enhanced detection of low-abundant proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with 2D-LC-MS/MS, including 42 proteins with normal reported concentrations ranging from ~100 pg/mL to 100 ng/mL. The concentrations of two selected proteins M-CSF and MMP8 were independently validated by ELISA as 202 pg/mL and 12.4 ng/mL, respectively. Evaluation of binding efficiency revealed 45 medium-abundant proteins efficiently captured by the SuperMix column with >90% retention. Taken together, the results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy in proteomic applications involving human biofluids where effectively addressing the dynamic range of the specimen is imperative.