ENRICHMENT AND DETECTION METHODOLOGIES FOR EGFR PHOSPHOPEPTIDES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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。目前，磷酸肽的富集法有多种，包括免疫沉淀法、强阳离子交换层析、静电排斥亲水作用层析、固定化金属亲和层析、二氧化锆层析和二氧化钛层析等。单独使用，这些方法中的每一种都确实提高了对磷酸肽的检测效率；每种方法都有优点和局限性。因此，结合不同的磷酸肽富集法，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，将人角膜缘上皮细胞系(HCLEs)培养成融合细胞，用EGF刺激，然后用8M尿素加磷酸酶和蛋白酶抑制剂进行裂解。对于定量的磷酸化分析，使用了稳定的同位素标记细胞。总蛋白平均为10 mg，经还原、烷基化和胰酶消化。多肽用SepPak C18反相色谱脱盐。用三种抗磷酸酪氨酸抗体的组合免疫沉淀磷酸酪氨酸肽。通过静电斥力亲水作用层析(ERLIC)或强阳离子交换层析(SCX)进一步分离。利用固定化金属亲和层析(IMAC)进一步富集组分，并使用通过Triversa Nanomate(Advion)与LTQ-Orbitrap MS(Thermo-Fisher)偶联的NanAcuity UPLC(Waters)进行LC/MSN分析。单独使用时，SCX层析分离样品，导致对磷酸肽的检测效率较低。SCX组分中相对较高数量的非磷多肽在检测方面优于磷酸多肽。进一步用IMAC层析法或TiO2层析法对SCX组分进行磷基浓缩，消除了大部分非磷肽，显着提高了磷酸肽的检测效率。在一项平行实验中，我们使用ERLIC对多肽进行分级，并直接通过LC/MS/MS分析得到的分级，仅使用ERLIC就可以显著提高磷酸肽的检测率。然而，由于与生物样品中的磷酸丝氨酸或磷酸苏氨酸相比，磷酸酪氨酸的化学计量比较低，它们的检测水平仍然是一个挑战。目前正在进行进一步的研究，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法，在SCX或ERLIC分级之前对磷酸酪氨酸肽进行分离。然后将流过的部分一分为二，并进一步进行分馏--一半接受SCX，另一半接受ERLIC。所有免疫沉淀肽、SCX和ERLIC组分在LC/MSN分析前用IMac进一步磷化。

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.