PROTEIN SIGNATURES OF LEUKEMIC CELLS

R21/R33应用程序的总体目标是开发和应用比较的“全基因组”蛋白质组学方法，以确定恶性细胞的蛋白质/多肽签名在多大程度上增强了从细胞遗传学和组织病理学分析中获得的信息。具体地说，我们将评估髓系白血病分子细胞遗传学异常和蛋白质表达之间的关系，并确定独特的蛋白质表达模式(例如，蛋白质/肽签名)是否可以预测治疗反应。二维(2-D)凝胶电泳法、飞行时间质谱仪(MALDI-TOF-MS)和纳升/分钟流速电喷雾质谱仪(ESI-MS)将被结合在一种新的方法中，以定位原代白血病细胞和核裂解物中的低丰度和高丰度蛋白质/肽。在R21的应用中，我们将建立方法学来优化临床标本的可重复蛋白质组采样。在R33的应用中，我们将采用两种平行的方法，基于根据等电点(PI)和质量对蛋白质进行二维凝胶分离，来研究临床白血病标本的蛋白质组。在一种方法中，我们将量化正常和白血病样本的细胞和核裂解物中差异表达的中丰度到高丰度的蛋白，并在R21应用程序中优化凝胶蛋白检测。在另一种方法中，我们将从临床标本中创建细胞和核裂解产物的整个2-D凝胶的肽图，以通过识别低丰度蛋白质来增加2-D凝胶上蛋白质测量的动态范围。这两种方法将被用来1)使用低丰度和高丰度蛋白质/肽图谱建立初发t(15；17)APL样本在诊断和复发时的蛋白质特征，以及2)确定APL在诊断时的蛋白质/肽特征预测治疗反应的程度。此外，我们将开始研究开发基于抗体的试剂，以测试与预后不良的APL相关的凝胶蛋白信号的临床潜力。我们预计，蛋白质/多肽签名将为与分子细胞遗传学异常相关的蛋白质的生理活性形式提供新的见解，增加对肿瘤表型调控因素的了解，并识别新的诊断和预测肿瘤标记物。

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.