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获得的信息的程度。 具体来说，我们将评估髓系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）预测治疗反应。 将二维凝胶电泳、飞行时间质谱（MALDI-TOF-MS）和纳升/分钟流速电喷雾质谱（ESI-MS）相结合，以一种新的方法对原发性白血病细胞和细胞核裂解物中的低丰度和高丰度蛋白/肽进行定位。 在R21应用中，我们将建立优化临床标本可重复蛋白质组采样的方法。 在R33的应用中，我们将采用两种平行的方法，根据等电点（pI）和质量的蛋白质的2-D凝胶分离的基础上，研究临床白血病标本的蛋白质组。 在一种方法中，我们将量化差异表达的中到高丰度蛋白质的细胞和细胞核裂解物从正常和白血病标本我们的样品处理和凝胶蛋白质检测中优化的R21应用。 在另一种方法中，我们将创建来自临床标本的细胞和核裂解物的整个2-D凝胶的肽图谱，以通过鉴定低丰度蛋白质来增加2-D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/肽图谱建立诊断和复发时原发性t（15;17）APL标本的蛋白特征，2）确定诊断时APL的蛋白/肽特征预测治疗反应的程度。 此外，我们将启动研究，以开发基于抗体的试剂，以测试与不良免疫APL相关的凝胶蛋白特征的临床潜力。 我们预计，蛋白质/肽的签名将提供新的见解与分子细胞遗传学畸变相关的蛋白质的生理活性形式，增加对肿瘤表型调节因子的理解，并确定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.