Targeted Isolation and Identification of Sialylated Glycoproteins in Cancer Tissues, Cells and Biofluids

糖蛋白占位于细胞表面和细胞膜中的蛋白质的大部分。 细胞外环境在癌变过程中，这些糖蛋白的正常功能， 细胞-细胞粘附的结合配体、细胞外基质分子、信号传导受体，以及 免疫细胞发生改变。转移性进展的许多步骤涉及重组 和细胞外基质中糖蛋白的降解。因此，聚糖和糖蛋白 抗原仍然包含大多数FDA批准的诊断性癌症生物标志物。我们集团 最近开发了多个N-连接聚糖成像质谱工作流程， 表征临床相关肿瘤组织、生物流体和细胞中的糖组，以及 通过免疫捕获单独靶向的糖蛋白。在这个建议中，应用新的点击 特异性靶向α 2，3唾液酸抗原的化学试剂，如sialyl刘易斯A/CA 19 -9， 唾液酸刘易斯X和sTRA，将用于改善肿瘤相关基因的分离和测序。 从FFPE组织、生物流体和细胞中提取糖肽。标准蛋白酶和新的ECM靶向 蛋白酶将用于分析。为了实现这一目标，提出了三个具体目标：1。 从临床FFPE癌组织中分离、鉴定和共定位唾液酸糖蛋白; 2. 分离和鉴定血浆癌症样品中的唾液酸糖蛋白; 3.分离和鉴定 高水平α 2，3唾液酸抗原癌细胞系中的唾液酸糖蛋白。的 开发的碳水化合物靶向点击捕获试剂可以直接应用于任何FFPE组织， 使用简单的化学反应来检测感兴趣的细胞或生物流体。开发的工具组合 本文将有助于鉴定相关细胞中的特定糖肽和糖蛋白种类， 癌症样本可以应用于癌症预防，进展， 预后和生物标志物应用。

Glycoproteins account for the majority of the proteins located at the cell surface and in the extracellular environment. During carcinogenesis the normal function of these glycoproteins as binding ligands for cell-cell adhesion, extracellular matrix molecules, signaling receptors, and immune cells becomes altered. Many of the steps in metastatic progression involve re-organization and degradation of the glycoproteins in the extracellular matrix. Hence, glycan and glycoprotein antigens still comprise the majority of FDA approved diagnostic cancer biomarkers. Our group has recently developed multiple N-linked glycan imaging mass spectrometry workflows to characterize the glycomes in clinically relevant tumor tissues, biofluids and cells, as well as individually targeted glycoproteins by immunocapture. In this proposal, application of a new click chemistry reagent specific for targeting of α2,3 sialic acid antigens, like sialyl Lewis A/CA19-9, sialyl Lewis X and sTRA, will be used to improve isolation and sequencing of tumor-associated glycopeptides from FFPE tissues, biofluids and cells. Standard proteases and new ECM-targeted proteases will be used in the analyses. To accomplish this, three Specific Aims are proposed: 1. Isolate, identify and co-localize sialic acid glycoproteins from clinical FFPE cancer tissues; 2. Isolate and identify sialic acid glycoproteins in plasma cancer samples; 3. Isolate and identify sialic acid glycoproteins in cancer cell lines with high levels of α2,3 sialic antigens. The carbohydrate-targeted click capture reagents developed can be applied directly to any FFPE tissue, cell or biofluid of interest using simple chemical reactions. The combination of tools developed herein will facilitate the identification of specific glycopeptide and glycoprotein species in relevant cancer samples which can be applied across the spectrum of cancer prevention, progression, prognosis, and biomarker applications.