Novel Approaches for Structural Determination of Cancer Stem Cell Glycans

描述（由申请人提供）：胰腺癌预计今年将在美国造成32,000人死亡。这种高死亡率主要是由于缺乏可靠的早期肿瘤检测方法，以及缺乏产生治愈的治疗选择。传统癌症治疗的一个并发症是，它们很容易忽略被称为干细胞的一小部分细胞，而干细胞已被证明与肿瘤的增殖能力有关。异常的蛋白糖基化与癌症的发生和发展有关。特别是，具有唾液酸和硫酸盐基团的酸性聚糖经常被改变。有趣的是，最近发现的胰腺癌干细胞的细胞表面标记物是糖蛋白。然而，由于与不稳定、异质和支链聚糖的结构测定相关的巨大分析挑战，产生新的诊断和治疗方法（包括癌症疫苗）所需的详细的癌症相关聚糖结构很少。质谱法（MS）可提供灵敏、准确的多糖分析方法。然而，酸性分子质谱的一个主要挑战是电离效率低。第二个挑战是确定糖的分支和特定的连接。此外，唾液酸和硫酸盐基团非常不稳定，进一步损害电离和使硫酸盐定位困难。该应用侧重于开发新的质谱方法，用于鉴定和结构表征胰腺癌干细胞（潜在的生物标志物）独特表达的聚糖。具体来说，将利用氧化锆和二氧化钛表面化学从复杂混合物中富集唾液化和硫酸化聚糖，从而大大提高纳米级正相液相色谱傅里叶变换离子回旋共振（FTICR）质谱的检测效率。对于这些聚糖的结构测定，我们将分别利用金属辅助电子捕获解离、电子脱离解离和真空紫外光解离。增加糖的交叉环解理，这提供了链接信息，因此可以确定分支结构，并确定硫酸盐的位置。

DESCRIPTION (provided by applicant): Pancreatic cancer is expected to cause >32,000 deaths in he United States this year. This high mortality is largely due to lack of reliable methods for early tumor detection, and lack of treatment options that produce a cure. One complication with traditional cancer treatments is that they can easily miss the small subset of cells, termed stem cells, which have been shown to be responsible for a tumor's ability to proliferate. Aberrant protein glycosylation is linked to the onset and progression of cancer. Particularly, acidic glycans with sialic acid and sulfate groups are often altered. Interestingly, recently identified cell surface markers of pancreatic cancer stem cells are glycoproteins. However, due to tremendous analytical challenges associated with structural determination of labile, heterogeneous and branched glycans, detailed cancer-associated glycan structures, which are required for generation of novel diagnostics and therapeutics, including cancer vaccines, are scarce. Mass spectrometry (MS) can provide sensitive and accurate glycan analysis. However, a major challenge in acidic molecule MS is low ionization efficiency. A second challenge is the determination of saccharide branching and specific linkage. Also, sialic acids and sulfate groups are extremely labile, further compromising ionization and rendering sulfate localization difficult. This application focuses on developing novel MS approaches for identifying and structurally characterizing glycans uniquely expressed by pancreatic cancer stem cells (potential biomarkers). Specifically, zirconia and titania surface chemistry will be utilized to enrich sialylated and sulfated glycans from complex mixtures, thereby greatly improving their detection by nano-scale normal phase liquid chromatography Fourier transform ion cyclotron resonance (FTICR) MS. For structural determination of these glycans, we will utilize metal-assisted electron capture dissociation, electron detachment dissociation, and vacuum ultraviolet photodissociation, respectively, to increase sugar cross-ring cleavage, which provides linkage information and therefore allows determination of branched structures, and to determine sulfate location. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a major death cause in the United States with a five-year survival rate of <5%. This research focuses on developing novel approaches for improved detection and structural determination of carbohydrates (sugar molecules) present on the surface of pancreatic cancer stem cells, that is, a subset of cells within a tumor that is responsible for its ability to grow and propagate. Carbohydrates are known to be altered in cancer and therefore constitute promising targets for cancer vaccine development.