DECODING CANCER SIGNATURE GLYCOFORMS OF SERUM TUMOR MARK

血清前列腺特异性抗原(PSA)水平的测量在监测已知前列腺癌患者中具有公认的作用。广泛使用PSA作为前列腺癌的筛查方法，虽然存在争议，但已导致癌症检测的增加和更早。广泛使用PSA作为前列腺癌的筛查方法，虽然存在争议，但已导致癌症检测的增加和更早。目前，迫切需要一种测试，以区分治疗的益处以及哪些患者可以避免不必要的激进治疗。在癌症糖生物学的基础研究中，有希望的新进展提供了一种合理的机制，通过这种机制，前列腺癌细胞表面的特定糖链改变为黏附分子，从而调节癌症的侵袭性。此外，已有报道在人类前列腺癌细胞系的PSA上存在这些糖链结构。这项应用的目标是开发和改进一种能够使用单个患者的血清进行高通量分析的PSA糖型分析方法。该方法的成功开发将实现三个特定目标：1)确定患者之间PSA糖形含量的差异，2)肿瘤标记物糖基化模式与患者临床状态之间的初步关联，3)阐明特定目标1和2中确定的PSA糖链结构的初始结构细节。具体目标将通过应用一种新的毛细管电泳法来检测PSA糖变异体，使用纳克或亚纳克糖蛋白。与传统的基于分辨率的糖型分析方法不同，这个试点项目将使用毛细管电泳峰模式迁移率测定来识别具有不同糖链的PSA分子。将进行毛细管电泳法分析，以便不分解组分糖形式，而是使每个个体根据其量和迁移率成比例地促进峰模式的迁移率。复制移动性确定允许进行统计分析。在流动性方面发现统计上的显著差异表明存在潜在的结构差异。通过特定的重组糖苷酶减少这种迁移率差异将为统计证实的迁移率差异的结构基础提供证据。与更标准的方法相比，这种方法的优势在于对流动性值精度的优化没有理论上的限制。不同PSA糖型之间可以分辨的结构细节程度取决于迁移率值测定的精确度。对迁移率值精度有重要影响的优化参数包括：毛细管壁平衡、样品基质控制、缓冲液稳定性、离子耗竭、样品蒸发、时间、时间分辨率、样品稳定性、PSA-丝氨酸络合、温度控制、样品进样、相同的塞子长度和样品浓度。优化后的分析方法将用于分析一系列预期的血清PSA样本，以确定唾液酸化和/或N-葡聚糖分支的显著变化。这些发现将与患者的临床状况相关。

Measurement of the serum level of prostate specific antigen (PSA) and a well-established role in monitoring patients with known prostate cancer. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Currently, there is an urgent need for a test that will distinguish benefit from treatment and which patients can avoid unnecessary aggressive therapies. Promising new developments in basic research on the glycobiology of cancer have provided a plausible mechanism by which specific glycan alterations to adhesion molecules on the surface of prostate cancer cells modulate the aggressiveness of the cancer. Furthermore, the presence of these glycan structures on PSA from a human prostate cancer cell line has been reported. The goal of this application is the development and refinement of a glycoform assay for PSA that is capable of high throughput analysis using serum from individual patients. Successful development of this assay will accomplishment of three specific aims: 1) determination of the patient-to- patient variation in glycoform content of PSA, 2) draw preliminary correlations between tumor marker glycosylation pattern and patient clinical status, and 3) elucidation of the initial structural detail of the PSA glycan structures identified in specific aims 1 and 2. The specific aims will be accomplished by applying a novel capillary electroporesis (CE) approach to detect PSA glycovariants using nanogram or subnanogram amounts of glycoprotein. In contrast to the traditional resolution based approach to glycoform analysis, this pilot project will use capillary electrophoresis peak mode mobility determinations to identify PSA molecules with variant glycans. CE analysis will be performed so that the component glycoforms are not resolved but so that each individual contributes to the mobility of the peak mode in proportion to its amount and mobility. Replicate mobility determinations allows statistical analysis. The identification of a statistically significant difference in mobility indicates an underlying structural difference. Reduction of that mobility difference by specific recombinant glycosidases will provide evidence of the structural basis of statistically confirmed mobility differences. The strength of this approach over more standard methodologies is that there is no theoretical limit to optimization of mobility value precision. The degree of structural detail that can be resolved between different PSA glycoforms is dependent on the precision of mobility value determinations. The parameters important to mobility value precision that will be optimized include:: capillary wall equilibration, sample matrix control, buffer stability, ion depletion, sample evaporation, temporal, temporal resolution, sample stability, PSA- serpin complexation, temperature control, sample injection, same plug length, and sample concentration. The optimized assay will be used to analyze a prospective series of serum PSA specimens for significant changes in sialylation and/or N-glycan branching. These findings will be correlated with the patient's clinical status.