Combining Protein-Based IMAC, Peptide-Based IMAC, and MudPIT for Efficient Phosphoproteomic Analysis

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.

固定化金属亲和色谱（Immobilized Metal Affinity Chromatography，IMAC）是从酶解蛋白质混合物中富集磷酸肽的常用策略。然而，单独使用该策略分析复杂蛋白质混合物时效率欠佳。本研究评估了以蛋白质为基础的IMAC作为肽基IMAC预富集步骤的应用效果。最终，我们将两种基于IMAC的富集方法与多维蛋白质鉴定技术（MudPIT）相结合，对哺乳动物细胞中的表皮生长因子通路开展定量磷酸化蛋白质组学分析，共鉴定出4470条独特磷酸肽，涵盖4729个磷酸化位点。