Discovery of Cancer Biomarkers Through Metabolic Engineering

描述（由申请人提供）：已知黏液蛋白o型连接糖基化的变化与癌症有关。然而，与黏液型糖基化相关的复杂性以及研究这种糖蛋白的有限工具严重限制了我们对其参与疾病的理解。寻找生物标记物正在成为基于质谱的蛋白质组学领域的一个主要焦点。然而，蛋白质表达水平的变化一直是该领域的主要焦点。最近，代谢工程和质谱技术已经发展到研究聚糖结构。这些技术为糖生物学家研究与生物标志物相关的聚糖结构变化带来了希望。我们的初步研究表明，通过Staudinger结扎或“点击化学”捕获代谢结合的o -连接叠氮糖，为使用质谱（MS）检测糖蛋白提供了一种方法。我们提出了一种基于质谱的方法来检测与培养细胞和小鼠肝癌模型中糖蛋白聚糖结构变化相关的癌症生物标志物。该提案的具体目标是：(1)开发一种从复杂裂解物中鉴定粘蛋白型糖基化的方法。(2)建立一种方法，通过稳定同位素标记和质谱法来量化复杂裂解物中蛋白质和糖基化水平的变化。(3)分析小鼠肝癌模型中黏蛋白型糖基化水平的变化。通过代谢工程将叠氮糖结合到糖蛋白中，然后通过“点击化学”与可切割亲和标签进行选择性化学反应，将为我们提供一种使用质谱法检测糖蛋白的方法，这些糖蛋白不一定是最丰富的蛋白质，而是那些在高通量下产生的蛋白质。使用具有独特特征的稳定同位素标记的炔基标签将使我们能够检测和量化糖基化和非糖基化肽，并比较来自培养细胞和小鼠肝癌模型trem - mycxlap - tta小鼠的健康和患病样本之间表达水平的差异。该项目将有助于我们从基础科学的角度理解粘蛋白型o链糖基化，以及发现癌症生物标志物。由于在几乎所有哺乳动物细胞的蛋白质上都发现了糖，因此在几乎任何类型的组织中都有可能发现癌症的生物标志物。发现可靠的生物标志物用于疾病的早期检测，无疑将提高诊断的准确性和患者的长期生存，并为药物开发提供治疗靶点。

DESCRIPTION (provided by applicant): Changes in mucin-type O-linked glycosylation of proteins are known to be linked to cancer. However, the complexity associated with mucin-type glycosylation and the limited tools available to study such glycoproteins has severely limited our understanding of their involvement in disease. The search for biomarkers is becoming a major focus in the field of mass spectrometry-based proteomics. However, changes in protein expression levels have been the main focus in this field. More recently, metabolic engineering and mass spectrometric techniques have been developed to study glycan structure. These techniques hold promise for glycobiologists studying changes to glycan structure related to biomarkers. Our preliminary studies have shown capture of metabolically incorporated O-linked azidosugars by Staudinger Ligation or "click chemistry" provides a method of detection for glycoproteins using mass spectrometry (MS). We propose a mass spectrometry-based method to detect biomarkers of cancer associated with changes to glycan structure of glycoproteins in cultured cells and a murine liver cancer model. Specific aims of the proposal are (1) Develop a method for identifying mucin-type glycosylation from complex lysates. (2) Develop a method to quantify changes in protein and glycosylation levels in complex lysates through stable isotope labeling and mass spectrometry. (3) Analyze changes in mucin-type glycosylation levels in a murine liver cancer model. Incorporating azidosugars into glycoproteins through metabolic engineering followed by a selective chemical reaction via "click chemistry" with a cleavable affinity tag will provide us with a method for the detection of glycoproteins using mass spectrometry that are not necessarily the most abundant proteins, but those that are produced at high flux. The use of a stable isotope labeled alkynyl tag with a unique signature will allow us to detect and quantify glycosylated and non-glycosylated peptides and compare differences in expression levels between healthy and diseased samples from cultured cells and a murine liver cancer model, the TRE-MYCxLAP-TTA mouse. The proposed project will contribute to our understanding of mucin-type O-linked glycosylation both from a basic science perspective as well as the discovery of cancer biomarkers. PUBLIC HEALTH RELEVANCE As sugars are found on proteins in nearly all mammalian cells, it is possible to find biomarkers of cancer in virtually any type of tissue. The discovery of reliable biomarkers for early detection of disease would undoubtedly improve the accuracy of diagnosis and long-term patient survival, and provide therapeutic targets for drug development.