ENRICHMENT AND DETECTION METHODOLOGIES FOR EGFR PHOSPHOPEPTIDES

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。 表皮生长因子受体(EGFR)酪氨酸激酶在调节细胞生长、增殖和迁移中起重要作用。EGFR中特定酪氨酸残基对不同外界刺激(如EGF、ATP、细胞损伤等)的差异磷酸化是这些刺激和它们激活的内部信号通路之间的关键纽带。EGFR亚磷酸盐特异性抗体一直被用作监测磷酸化位点占有率的高度敏感的工具，然而，它们缺乏特异性。最近，基于高灵敏质谱学的检测策略被描述为在各种条件下表征EGFR酪氨酸磷酸化级联反应。我们正在探索优化样品制备、磷酸肽的富集化和检测策略，以研究EGFR磷酸肽。将高表达EGFR的人表皮样癌A431细胞、转EGFR表达载体的猪主动脉内皮细胞系或小鼠角膜上皮细胞在体外培养至融合生长，在含有蛋白水解酶和磷酸酶抑制剂的混合物中收集细胞，并对EGFR进行免疫沉淀。洗脱的EGFR，或对照，商业上可获得的纯化的EGFR(来自A431细胞)经过SDS-PAGE和一组蛋白酶的凝胶内消化。多肽通过反相、离子交换、金属离子亲和层析或二氧化钛亲和层析进行洗脱和浓缩。使用多种基质和基质添加剂，在正离子和负离子模式下用MALDI-TOF MS分析了磷肽。我们用免疫沉淀和SDS-PAGE从培养的细胞中高产率和高纯度地回收了EGFR。我们已经探索了使用多种酶进行凝胶内消化，以定位，特别是大的含有酪氨酸的胰蛋白酶多肽，这些多肽可能在以前的MS分析中没有出现。使用优化的凝胶回收程序，我们对EGFR多肽进行了各种形式的色谱浓缩和分离技术，以利用EGFR磷酸肽的差异结合和洗脱。一组基质和基质添加剂正在探索它们在MALDI-TOF MS的正离子和负离子模式下增强EGFR磷酸肽的电离能力。初步结果表明，为了最大限度地检测EGFR磷酸肽，可以在样品制备、浓缩和电离方法方面做出重大改进。

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. Epidermal growth factor receptor (EGFR) tyrosine kinase plays an important role in regulating cell growth, proliferation, and migration. Differential phosphorylation of specific tyrosine residues in EGFR in response to diverse external stimuli (e.g., exposure to EGF, ATP, cell injury, etc.) serves as the key link between these stimuli and the internal signaling pathways that they activate. EGFR phosphosite-specific antibodies have been used as highly sensitive tools to monitor phosphorylation site occupancy, however, they suffer from a lack of specificity. Recently, highly sensitive mass spectrometric-based detection strategies have been described to characterize the EGFR tyrosine phosphorylation cascade under a variety of conditions. We are exploring the optimization of sample preparation, phosphopeptide enrichment and detection strategies for the study of EGFR phosphopeptides. Human epidermoid carcinoma A431 cells, which over-express EGFR, or the porcine aortic endothelial (PAE) cell line transfected with EGFR expression constructs, or mouse corneal epithelial cells, were grown in culture to confluence, harvested in the presence of a cocktail of protease and phosphatase inhibitors, and EGFR was subjected to immunoprecipitation. Eluted EGFR, or control, commercially-available purified EGFR (derived from A431 cells) was subjected to SDS-PAGE and in-gel digestion with a panel of proteases. Peptides were eluted and subjected to enrichment by reversed-phase, ion exchange, metal ion affinity, or titanium dioxide affinity chromatography. Phosphopeptides were analyzed by MALDI-TOF MS using a variety of matrices and matrix additives in the positive and negative ion modes. We have recovered EGFR from cells in culture with high yield and purity using immunoprecipitation followed by SDS-PAGE. We have explored the use of multiple proteases for in-gel digestion, to target, in particular large tyrosine-containing tryptic peptides that may have been unrepresented in previous MS analyses. Using optimized procedures for recovery from gel, we have subjected the EGFR peptides to various forms of chromatographic enrichment and separation techniques to exploit the differential binding and elution of EGFR phosphopeptides. A panel of matrices and matrix additives are being explored for their capacity to enhance ionization of EGFR phosphopeptides in the positive and negative ion mode of the MALDI-TOF MS. Initial results suggest that significant improvements can be made in sample preparation, enrichment and ionization methodologies in order to maximize the detection of EGFR phosphopeptides.