ENRICHMENT AND DETECTION METHODOLOGIES FOR EGFR PHOSPHOPEPTIDES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 表皮生长因子受体（EGFR）酪氨酸激酶在调节细胞生长、增殖和迁移方面发挥着重要作用。EGFR中特定酪氨酸残基响应于不同外部刺激（例如，EGF、ATP、细胞损伤等）作为这些刺激和它们激活的内部信号通路之间的关键环节。EGFR磷酸位点特异性抗体已被用作监测磷酸化位点占用的高度敏感的工具，然而，它们缺乏特异性。最近，高灵敏度的基于质谱的检测策略已被描述为在各种条件下表征EGFR酪氨酸磷酸化级联。我们正在探索EGFR磷酸肽研究的样品制备、磷酸肽富集和检测策略的优化。 将过表达EGFR的人表皮样癌A431细胞、或用EGFR表达构建体转染的猪主动脉内皮（PAE）细胞系、或小鼠角膜上皮细胞在培养物中生长至汇合，在蛋白酶和磷酸酶抑制剂的混合物存在下收获，并对EGFR进行免疫沉淀。将洗脱的EGFR或对照、市售纯化的EGFR（来源于A431细胞）进行SDS-PAGE并用一组蛋白酶进行凝胶内消化。肽被洗脱，并通过反相、离子交换、金属离子亲和或二氧化钛亲和色谱进行富集。通过MALDI-TOF MS使用各种基质和基质添加剂以正离子和负离子模式分析磷酸肽。我们已经从培养的细胞中回收了EGFR，使用免疫沉淀，然后进行SDS-PAGE，具有高产量和纯度。我们已经探索了使用多种蛋白酶进行凝胶内消化，以靶向，特别是可能在先前的MS分析中未被代表的含酪氨酸的胰蛋白酶肽。使用优化的程序从凝胶中回收，我们已经进行了EGFR肽的各种形式的色谱富集和分离技术，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富集方案，然后使用Bruker Reflex IV进行MALDI-TOF MS肽图谱分析，并使用与Advion Nanomate和Thermo-Fisher LTQ-Orbitrap MS连接的沃茨Acuity NanoLC进行nanoLC/MSn分析，我们已经能够建立EGFR响应损伤以及UTP或EGF刺激的差异磷酸化。与EGF直接刺激E1 PAE细胞相比，UTP刺激后EGFR的磷酸化迅速减弱。EGFR磷酸化的强度被发现显着降低时，细胞受到刺激的UTP，而不是刺激的EGF。 我们观察到Grb 2结合随着时间的推移逐渐增加，并在EGF刺激后45分钟达到峰值，此后迅速减弱。酪氨酸残基1068和1086突变为苯丙氨酸减少Grb 2的结合时，细胞与UTP刺激，但观察到一个较小的减少与EGF刺激。我们还发现，无论是损伤还是UTP，但EGF招募Grb 2 EGFR。我们的数据表明，这是由于EGFR酪氨酸残基的差异磷酸化，结果与不同的激动剂刺激。ITRAQ和SILAC方法现在被用来量化EGFR位点的磷酸化，并确定在该过程中作为相互作用的parthers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.