Quantitative Serum Proteomics from Surface Plasmon Resonance Imaging
Quantitative Serum Proteomics from Surface Plasmon Resonance Imaging
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DOI：
10.1074/mcp.m800121-mcp200
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发表时间：
2008-12-01
影响因子：
7
通讯作者：
Hood, Leroy
中科院分区：
文献类型：
--
作者：
Lausted, Christopher;Hu, Zhiyuan;Hood, Leroy
复杂混合物中特定蛋白质的检测和定量是蛋白质组学面临的主要挑战。例如，开发与疾病相关的生物标志物小组将需要快速有效的方法来获得多参数蛋白质图谱。我们建立了一种利用抗体微阵列的表面等离子体共振成像来分析血清的高通量、无标记的方法。在裸金衬底上使用标准的针点法制作微阵列，并使用基于相机的表面等离子体共振系统将样品应用于结合分析。我们通过使用792特征微阵列的一部分来测量四种血清蛋白质的浓度来验证该系统。人血清和小鼠血清中的转铁蛋白浓度分别为2.1 mg/ml和1.2 mg/ml，与ELISA法测定的2.6 mg/ml和1.2 mg/ml相近。人和小鼠的白蛋白水平分别为24.3和23.6 mg/ml，与预期值一致。四种测定值的最低检出限分别为14至58毫升或175至755微微米。在纯化的目标蛋白不能用于校准的情况下，微阵列可用于相对蛋白质定量。我们使用抗体芯片比较了三名肝癌患者和三名非肝癌患者的血清蛋白质谱。血清蛋白水平的等级聚类清楚地区分了两种不同的特征。检测到39个显著的蛋白质变化(p<；0.05)，其中10个以前在血清中观察到。甲胎蛋白，一种已知的肝癌标记物，被观察到增加。这些结果证明了这种高通量方法用于绝对和相对蛋白质表达谱分析的可行性。《分子与细胞蛋白质组学》7：2464-2474,2008。
The detection and quantification of specific proteins in complex mixtures is a major challenge for proteomics. For example, the development of disease-related biomarker panels will require fast and efficient methods for obtaining multiparameter protein profiles. We established a high throughput, label-free method for analyzing serum using surface plasmon resonance imaging of antibody microarrays. Microarrays were fabricated using standard pin spotting on bare gold substrates, and samples were applied for binding analysis using a camera-based surface plasmon resonance system. We validated the system by measuring the concentrations of four serum proteins using part of a 792-feature microarray. Transferrin concentrations were measured to be 2.1 mg/ml in human serum and 1.2 mg/ml in murine serum, which closely matched ELISA determinations of 2.6 and 1.2 mg/ml, respectively. In agreement with expected values, human and mouse albumin levels were measured to be 24.3 and 23.6 mg/ml, respectively. The lower limits of detection for the four measurements ranged from 14 to 58 ng/ml or 175 to 755 pM. Where purified target proteins are not available for calibration, the microarrays can be used for relative protein quantification. We used the antibody microarray to compare the serum protein profiles from three liver cancer patients and three non-liver cancer patients. Hierarchical clustering of the serum protein levels clearly distinguished two distinct profiles. Thirty-nine significant protein changes were detected (p < 0.05), 10 of which have been observed previously in serum. alpha-Fetoprotein, a known liver cancer marker, was observed to increase. These results demonstrate the feasibility of this high throughput approach for both absolute and relative protein expression profiling. Molecular & Cellular Proteomics 7:2464-2474, 2008.