Improved Two-Dimensional Reversed Phase-Reversed Phase LC-MS/MS Approach for Identification of Peptide-Protein Interactions

Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RP-RP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis.

定量质谱分析（MS）与亲和纯化技术的结合，允许可偏见的蛋白质-蛋白质及肽-蛋白质相互作用研究。在基于蛋白酶解复杂蛋白质混合物二维液相色谱的散弹法中，MS分析前的分离努力集中于肽段。本研究开发了一种改进的离线二维液相色谱-质谱/质谱（LC-MS/MS）方法，通过使用反相色谱毛细管柱和酸性乙腈含溶质在色谱两维中的洗脱剂，以实现对结合蛋白的鉴定和定量。采用特定的分级方案，以获取肽段分布均匀的样本，并充分利用第二维纳米液相色谱-质谱/质谱的分离空间。我们报道了肽-蛋白质相互作用研究，以鉴定T细胞适配蛋白ADAP的磷酸化依赖性结合伙伴。基于SILAC的拉下实验结果表明，此方法非常适合区分磷酸化特异性相互作用与非特异性结合事件。数据进一步证实，ADAP中磷酸化的酪氨酸595可能作为T细胞蛋白SLP76和NCK的SH2结构域的直接结合位点。从技术角度来看，我们提供了一种离线二维反相色谱-反相色谱LC-MS/MS方法的详细方案，该方法为定量相互作用组分析提供了一种稳健且节省时间的替代方案。