COMBINING PROTEIN-BASED IMAC, PEPTIDE-BASED IMAC, AND MUDPIT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 固定化金属亲和层析(IMAC)是一种常用的从消化的蛋白质混合物中富集磷酸肽的方法。然而，在分析复杂的蛋白质混合物时，这种策略本身是低效的。在这里，我们评估了在使用基于多肽的IMAC之前，使用基于蛋白质的IMAC作为预浓缩步骤的有效性。最终，我们将这两个基于iMac的富集物和MudPIT结合在一起，对哺乳动物细胞中的表皮生长因子途径进行了定量的磷酸蛋白质组学分析，确定了4470个独特的磷酸化多肽，包含4729个磷酸化位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.