PROTEIN SIGNATURES OF LEUKEMIC CELLS

该 R21/R33 应用的总体目标是开发和应用比较“全基因组”蛋白质组学方法，以确定恶性细胞的蛋白质/肽特征增强从细胞遗传学和组织病理学分析中获得的信息的程度。 具体来说，我们将评估骨髓性白血病中分子细胞遗传学畸变和蛋白质表达之间的关系，并确定蛋白质表达的独特模式（例如蛋白质/肽特征）是否可以预测治疗反应。 二维 (2-D) 凝胶电泳、飞行时间质谱 (MALDI-TOF-MS) 和纳升/分钟流速电喷雾质谱 (ESI-MS) 将结合在一种新方法中，以绘制原代白血病样本细胞和核裂解物中低丰度和高丰度蛋白质/肽的图谱。 在 R21 应用中，我们将建立方法来优化临床样本的可重复蛋白质组采样。 在 R33 应用中，我们将采用两种并行方法，基于根据等电点 (pI) 和质量对蛋白质进行二维凝胶分离，以研究临床白血病样本的蛋白质组。 在一种方法中，我们将通过在 R21 应用程序中优化的样品处理和凝胶蛋白检测来定量正常和白血病样本的细胞和细胞核裂解物中差异表达的中等到高丰度蛋白质。 在另一种方法中，我们将创建临床样本中细胞和核裂解物的整个二维凝胶的肽图，通过识别低丰度蛋白质来增加二维凝胶上蛋白质测量的动态范围。 这两种方法都将用于 1) 使用低丰度和高丰度蛋白质/肽图谱建立原发 t(15;17) APL 样本在诊断和复发时的蛋白质特征，2) 确定诊断时 APL 的蛋白质/肽特征预测治疗反应的程度。 此外，我们将启动研究开发基于抗体的试剂，以测试与不良预后 APL 相关的凝胶蛋白特征的临床潜力。 我们预计蛋白质/肽特征将为与分子细胞遗传学畸变相关的蛋白质的生理活性形式提供新的见解，增加对肿瘤表型调节因子的了解，并识别新的诊断和预测肿瘤标志物。

The overall goal of this R21/R33 application is to develop and apply comparative "genome-wide" proteomic approaches to determine the extent to which protein/peptide signatures of malignant cells enhance information obtained from cytogenetic and histopathologic analyses. Specifically, we will evaluate the relationship between molecular cytogenetic aberrations and protein expression in myeloid leukemia and determine whether distinctive patterns of protein expression (e.g., protein/peptide signatures) predict treatment response. Two-dimensional (2-D) gel electrophoresis, time-of-flight mass spectrometry (MALDI-TOF-MS) and nanoliter/min flow rate electrospray mass spectromety (ESI-MS) will be combined in a novel approach to map low and high abundance proteins/peptides in cell and nuclear lysates from primary leukemic specimens. In the R21 application we will establish methodologies to optimize reproducible proteome sampling of clinical specimens. In the R33 application, we will pursue two parallel approaches, based on 2-D gel separation of proteins according to isoelectric points (pI) and mass, to investigate the proteome of clinical leukemic specimens. In one approach, we will quantify differentially expressed moderate-to high abundance proteins in cell and nuclei lysates from normal and leukemic specimens us sample processing and in gel protein detection optimized in the R21 application. In another approach we will create peptide maps of the entire 2-D gel of cell and nuclear lysates from clinical specimens to increase the dynamic range of protein measurements on 2-D gels by identifying low abundance proteins. Both of these approaches will be used to 1) establish the protein signature of primary t(15;17) APL specimens at diagnosis and relapse using low and high abundance protein/peptide maps and 2) determine the extent to which protein/peptide signatures of APL at diagnosis predict treatment response. Additionally, we will initiate studies to develop antibody-based reagents to test the clinical potential of the gel protein signature associated with poor prognoses APL. We anticipate that protein/peptide signatures will lend new insight into the physiologically active forms of proteins associated with molecular cytogenetic aberrations, increase understanding about regulators of tumor phenotype, and identify novel diagnostic and predictive tumor markers.