Toward a human blood serum proteome - Analysis by multidimensional separation coupled with mass spectrometry
Toward a human blood serum proteome - Analysis by multidimensional separation coupled with mass spectrometry
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DOI：
10.1074/mcp.m200066-mcp200
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发表时间：
2002-12-01
影响因子：
7
通讯作者：
Pounds, JG
中科院分区：
文献类型：
--
作者：
Adkins, JN;Varnum, SM;Pounds, JG
血清是一种复杂的体液，含有浓度范围至少超过9个数量级的各种蛋白质。使用质谱技术与样品制备的改进相结合，我们用亚毫升量的血清进行了蛋白质组学分析，并增加了血清中蛋白质的可测量浓度范围，超出了以前的报告。采用在线反相微毛细管液相色谱-离子阱质谱联用技术，对血清中490种蛋白质进行了检测。为了进行该分析，使用蛋白A/G从血清中除去免疫球蛋白，并使用胰蛋白酶消化剩余的蛋白。在分析之前，通过强阳离子交换色谱将所得肽分离成不同的级分。这种分离导致在单个血清样品中检测到的蛋白质数量增加3-5倍。随着鉴定的蛋白质数量的增加，我们检测到了一些丰度较低的血清蛋白（ng/ml范围），包括人生长激素、白细胞介素-12和前列腺特异性抗原。我们还使用SEQUEST来比较不同的蛋白质数据库，有和没有过滤。绘制这种比较，以允许快速视觉评估不同的数据库作为分析质量的主观衡量。通过这项研究，我们对血清蛋白进行了迄今为止最广泛的分析，并为未来改进识别疾病的新型蛋白质生物标志物奠定了基础。
Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.