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.
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机译：在通过IgY亲和力色谱柱分离出高度丰富的蛋白质（HAP）之后，下一层丰富的蛋白质，即中度丰富的蛋白质（MAP），成为获取低丰富的蛋白质（LAP）的障碍，在这里，大多数生物学相关且在临床上很重要的蛋白质生物标记物驻留。因此，MAP的隔离是有效检测和分析LAP的新挑战。为了应对这一挑战，我们通过用IgY12色谱柱的流通部分免疫鸡，并用针对IgY12色谱柱的流通蛋白的亲和纯化的IgY抗体构建了该柱，从而进一步开发了IgY-微珠系统。所开发的称为SuperMix的色谱柱用于进一步分离IgY12的流通级分，从而得到结合/洗脱级分（称为MAP级分）和流通级分（称为LAP级分）。通过SDS-PAGE和2DE对使用IgY12和SuperMix色谱柱进行的未分级分离和连续级分离的样品进行了分析。我们的数据表明，SuperMix色谱柱可特异性地，可重复地去除丰富蛋白质的IgY12后层。使用SDS-PAGE结合LC / MS / MS进行的案例研究表明，SuperMix色谱柱可实现207 MAP的特异性捕获，并以高可信度（≥95％）唯一鉴定出77种蛋白质。这种新颖的方法可以更深入，更有效地访问LAP群体。除了使用SuperMix色谱柱进行更深入的研究外，我们还朝着更快的方向发展。血浆生物标志物发现的当前挑战之一是样品通量限制。与PSS Bio Instruments合作，GenWay Biotech开发了一种新颖的多重自动化系统（SepproTip），用于高通量血浆样品处理。它允许一次处理12个样本。此Seppro-Tip系统可以使用IgY12和SuperMix吸头处理血浆样品。每65分钟12个样品的周转时间可以处理大量样品，而不会降低样品制备质量。
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