ENRICHMENT AND DETECTION METHODOLOGIES FOR EGFR PHOSPHOPEPTIDES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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。 目前，许多磷酸肽富集方法正在使用中，包括磷蛋白/肽与特异性抗体的免疫沉淀、强阳离子交换色谱、静电排斥亲水相互作用色谱、固定化金属亲和色谱、二氧化锆和二氧化钛色谱。单独使用，这些方法中的每一种都提高了磷酸肽的检测效率;每一种都有优点和局限性。因此，结合不同的磷酸肽富集方法，对于更全面地检测复杂生物样品中的磷酸肽至关重要。 在我们最近的实验中，人角膜缘上皮细胞系（HCLE）生长至汇合，用EGF刺激，并用补充有磷酸酶和蛋白酶抑制剂的8 M尿素裂解。对于定量磷酸化分析，使用稳定同位素标记的细胞。平均10 mg总蛋白被还原、烷基化并用胰蛋白酶消化。肽用SepPak C18反相色谱脱盐。使用三种抗磷酸酪氨酸抗体的组合免疫沉淀磷酸酪氨酸肽。用静电排斥亲水相互作用色谱（ERLIC）或强阳离子交换（SCX）色谱进一步分级流出物。使用固定化金属亲和色谱法（IMAC）进一步磷酸富集级分，并使用通过TriVersa NanoMate（Advion）偶联至LTQ-Orbitrap MS（Thermo-Fisher）的nanoAcuity UPLC（沃茨）通过LC/MSn进行分析。 当单独使用时，样品的SCX色谱分离导致磷酸肽的检测效率低。SCX级分中相对高量的非磷酸肽胜过磷酸肽用于检测。进一步磷酸基富集的SCX馏分与IMAC或TiO 2色谱消除了大部分的非磷酸肽和显着增加的磷酸肽的检测效率。在一个平行的实验中，我们分馏肽使用ERLIC和分析直接通过LC/MS/MS得到的馏分。ERLIC单独提供显着增加检测磷酸肽。然而，鉴于生物样品中磷酸酪氨酸与磷酸丝氨酸或磷酸苏氨酸相比化学计量较低，它们的检测水平仍然是一个挑战。目前正在进一步研究以提高磷酸酪氨酸肽的检测效率。我们目前的方法采用免疫沉淀的磷酸酪氨酸肽之前，通过SCX或ERLIC分级。然后将流出物分成两部分并进一步分馏-一半进行SCX，另一半进行ERLIC。在LC/MSn分析之前，使用IMAC进一步磷酸化富集所有免疫沉淀的肽、SCX和ERLIC级分。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Currently, many phosphopeptide enrichment methods are in use, including immunoprecipitation of phosphoproteins/peptides with specific antibodies, strong cation exchange chromatography, electrostatic repulsion hydrophilic interaction chromatography, immobilized metal affinity chromatography, zirconium dioxide and titanium dioxide chromatography. Used independently, each of these methods does increase the efficiency of detection for phosphopeptides;each has advantages and limitations. Combining different phosphopeptide enrichment methods is, therefore, crucial for a more comprehensive detection of phosphopeptides in complex biological samples. In our most recent experiments, Human Corneal Limbal Epithelial cell lines (HCLEs) were grown to confluence, stimulated with EGF, and lysed with 8M urea supplemented with phosphatase and protease inhibitors. For quantitative phosphorylation analyses, stable isotope-labeled cells were used. An average of 10 mg total protein was reduced, alkylated and digested with trypsin. Peptides were desalted with SepPak C18 reversed phase chromatography. Phosphotyrosine peptides were immunoprecipitated using a combination of three anti-phosphotyrosine antibodies. The flow-through was further fractionated with either electrostatic repulsion hydrophilic interaction chromatography (ERLIC) or strong cation exchange (SCX) chromatography. Fractions were further phosphoenriched using immobilized metal affinity chromatography (IMAC) and analyzed by LC/MSn using a nanoAcuity UPLC (Waters) coupled through a TriVersa NanoMate (Advion) to an LTQ-Orbitrap MS (Thermo-Fisher). When employed alone, SCX chromatographic fractionation of samples, resulted in low detection efficiency for phosphopeptides. The relatively high amount of non-phosphopeptides in the SCX fractions outcompeted the phosphopeptides for detection. Further phospho-based enrichment of the SCX fractions with either IMAC or TiO2 chromatography eliminated most of the non-phosphopeptides and significantly increased detection efficiency for the phosphopeptides. In a parallel experiment, we fractionated peptides using ERLIC and analyzed the resulting fractions directly by LC/MS/MS. ERLIC alone provided significantly increased detection of phosphopeptides. Given the low stoichiometry of phosphotyrosine compared to phosphoserine or phosphothreonine in biological samples, however, their detection level of remains a challenge. Further reasearch is presently underway to increase the detection efficiency for phosphotyrosine peptides. Our current approach employs immunoprecipitation of phosphotyrosine peptides prior to fractionation by either SCX or ERLIC. The flow-through is then divided in to two and fractionated further - one half is subjected to SCX and the other half to ERLIC. All the immunoprecipitated peptides, the SCX and ERLIC fractions are further phosphoenriched using IMAC before LC/MSn analysis.