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。使用优化的磷酸肽回收和浓缩程序，然后使用Bruker Reflex IV的MALDI-TOF MS肽图和Waters Acuity NanoLC与Advion Nanomate和Thermo-Fisher LTQ-Orbitrap MS的NanoLC/MSN分析，我们已经能够建立EGFR在损伤、UTP或EGF刺激下的差异磷酸化。与EGF直接刺激E1PAE细胞相比，UTP刺激后EGFR的磷酸化迅速减弱。UTP刺激细胞时，EGFR的磷酸化强度显著降低，而EGF刺激时则相反。我们观察到Grb2结合随着时间的推移逐渐增加，并在EGF刺激后45min达到峰值，此后迅速减弱。当细胞受到UTP刺激时，酪氨酸残基1068和1086突变为苯丙氨酸，减少了Grb2的结合，但在EGF刺激下，减少的幅度较小。我们还发现，既不是损伤也不是UTP，但EGF将Grb2招募到EGFR。我们的数据表明，这是由于不同激动剂刺激导致的EGFR酪氨酸残基的差异磷酸化。ITRAQ和SILAC方法现在被用来量化EGFR位点的磷酸化，并确定在这一过程中充当相互作用伙伴的其他蛋白质。

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. Using an optimized phosphopeptide recovery and enrichment protocol, followed by MALDI-TOF MS peptide mapping with a Bruker Reflex IV and nanoLC/MSn analysis with a Waters Acuity NanoLC interfaced to an Advion Nanomate and Thermo-Fisher LTQ-Orbitrap MS, we have been able to establish the differential phosphorylation of EGFR in response to injury, and to UTP or EGF stimulation. Phosphorylation of EGFR following UTP stimulation attenuates rapidly compared to direct stimulation of E1PAE cells by EGF. The intensity of EGFR phosphorylation was found to be significantly reduced when cells were stimulated by UTP, as opposed to stimulation by EGF. We observed that Grb2 binding gradually increases over time and reaches its peak at 45 min post-EGF stimulation and attenuates rapidly thereafter. Mutation of tyrosine residues 1068 and 1086 to phenylalanine reduced Grb2 binding when cells were stimulated with UTP, but a smaller decrease was observed when stimulated with EGF. We have also found that neither injury nor UTP, but EGF recruits Grb2 to EGFR. Our data indicates that this is due to the differential phosphorylation of EGFR tyrosine residues that results from stimulation with different agonists. ITRAQ and SILAC methods are now being used to quantify the phosphorylation of EGFR sites and to determine other proteins that serve as interacting parthers in the process.