P28-S Digging Deeper and Faster into Proteome by IgY-Immunoaffinity Fractionation.

After separating the highly abundant proteins (HAP) by IgY affinity column, the next layer of abundant protein, moderately abundant proteins (MAP), becomes an obstacle to access the low abundant proteins (LAP), where the majority of biologically relevant and clinically important biomarkers reside. Therefore, isolation of MAP is a new challenge for effective detection and analysis of LAPs. To tackle this challenge, we further developed the IgY-microbead system by immunizing chickens with a flow-through fraction of IgY12 column and constructing the column with affinity-purified IgY antibodies against the flow-through proteins of IgY12 column. The column developed, called SuperMix, was applied for further partitioning of the flow-through fraction of IgY12, which resulted in a bound/eluted fraction (designated as MAP fraction) and the flow-through fraction (designated as LAP fraction). Unfractionated and serial-fractionated samples using IgY12 and SuperMix columns were analyzed by SDS-PAGE and 2DE. Our data demonstrate that SuperMix columns specifically and reproducibly remove the post-IgY12 layer of the abundant proteins. A case study using SDS-PAGE coupled with LC/MS/MS demonstrates that the SuperMix column enabled specific capturing of 207 MAP, with 77 proteins being uniquely identified in high confidence (≥95%). This novel approach enables deeper and more effective access into the population of LAPs. In addition to digging deeper with the SuperMix column, we also have progressed in the direction of digging faster. One of the present challenges of plasma biomarker discovery is sample throughput limitations. In collaboration with PSS Bio Instruments, GenWay Biotech has developed a novel multiplex automated system (SepproTip) for high-throughput plasma sample processing. It permits processing of 12 samples at a time. This Seppro-Tip system can process plasma samples using both IgY12 and SuperMix tips. The turnaround time of 12 samples per 65 min allows a large number of samples to be processed without decrease in sample preparation quality.