Ong Shao-En
The Broad Institute of MIT and Harvard, Cambridge MA 02142, USA.
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.prot5400. doi: 10.1101/pdb.prot5400.
The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein-bait interactions that drive biological processes. However, such experiments are often analyzed by Western blotting, limiting the ability to detect novel protein interactors. Unbiased protein identification by mass spectrometry (MS) extends these experiments beyond the study of pairwise interactions, allowing analyses of whole networks of protein-bait interactions. With the latest advances in MS, it is not uncommon to identify thousands of proteins from complex mixtures. Paradoxically, the improved sensitivity of proteomic analyses can make it more difficult to distinguish bait-specific interactions from the large background of identified proteins. In quantitative proteomics, MS signals from protein populations labeled with stable isotopes such as (13)C and (15)N can be identified and quantified relative to unlabeled counterparts. Using quantitative proteomics to compare biochemical enrichments with the bait of interest against those obtained with control baits allows sensitive detection and discrimination of specific protein-bait interactions among the large number of nonspecific interactions with beads. Ad hoc optimization of enrichment conditions is minimized, and mild purification conditions preserve secondary or high-order protein-protein interactions. The combination of biochemical enrichment and quantitative proteomics allows rapid characterization of molecular baits with their interacting proteins, providing tremendous insight into their biological mechanisms of action.
使用重组蛋白、抗体、小分子或核酸作为亲和试剂是研究驱动生物过程的蛋白质-诱饵相互作用的一种简单而强大的策略。然而，此类实验通常通过蛋白质印迹法进行分析，这限制了检测新型蛋白质相互作用分子的能力。通过质谱（MS）进行无偏倚的蛋白质鉴定将这些实验扩展到成对相互作用研究之外，允许对蛋白质-诱饵相互作用的整个网络进行分析。随着MS的最新进展，从复杂混合物中鉴定出数千种蛋白质并不罕见。矛盾的是，蛋白质组学分析灵敏度的提高可能会使区分诱饵特异性相互作用与大量已鉴定蛋白质的背景变得更加困难。在定量蛋白质组学中，可以相对于未标记的对应物鉴定和定量来自用稳定同位素如（13）C和（15）N标记的蛋白质群体的MS信号。使用定量蛋白质组学将感兴趣的诱饵的生化富集与对照诱饵获得的生化富集进行比较，可以在与珠子的大量非特异性相互作用中灵敏地检测和区分特定的蛋白质-诱饵相互作用。富集条件的临时优化被最小化，温和的纯化条件保留了二级或高阶蛋白质-蛋白质相互作用。生化富集和定量蛋白质组学的结合允许快速表征分子诱饵及其相互作用的蛋白质，从而深入了解它们的生物学作用机制。
