Peking University People's Hospital, Peking University Institute of Hematology, Beijing, China.
Mol Cell Biochem. 2010 Oct;343(1-2):91-9. doi: 10.1007/s11010-010-0502-x. Epub 2010 Jun 5.
Plasma is recognized as a promising source of disease-related biomarkers, and proteomic approaches for identifying novel plasma biomarkers are in great demand. However, the complexity and dynamic protein concentration range of plasma remain the main obstacles for current research in this field. In this study, plasma proteins were prefractioned by immunodepletion and Protein Equalizer Technology to remove high abundant proteins, then labeled with an 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) to improve the peptide ionization, and analyzed by strong-cation-exchange(SCX) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that both prefraction methods were complementary, with regard to the number of identified proteins. Good chromatographic technique is important to further fractionate the iTRAQ labeling peptides, which allowed 320 and 248 different proteins to be characterized from two prefraction methods, respectively, encompassing a wide array of biological functions and a broad dynamic range of 10(7). Furthermore, the accuracy of iTRAQ relative quantitation for differentially expressed proteins is associated with the number of peptides hits per protein.
血浆被认为是疾病相关生物标志物的一个很有前途的来源，因此，寻找鉴定新型血浆生物标志物的蛋白质组学方法受到了广泛的关注。然而，血浆的复杂性和动态蛋白浓度范围仍然是该领域当前研究的主要障碍。在本研究中，通过免疫沉淀和蛋白等量标记技术（Protein Equalizer Technology）对血浆蛋白进行预分级，以去除高丰度蛋白，然后用 8 重同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation，iTRAQ）试剂标记，以提高肽的离子化效率，并通过强阳离子交换（strong-cation-exchange，SCX）与液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry，LC-MS/MS）联用进行分析。我们的结果表明，这两种预分级方法在鉴定蛋白的数量上具有互补性。良好的色谱技术对于进一步分离 iTRAQ 标记肽非常重要，这使得两种预分级方法分别能够鉴定 320 种和 248 种不同的蛋白质，涵盖了广泛的生物学功能和 107 的动态范围。此外，iTRAQ 相对定量分析差异表达蛋白的准确性与每个蛋白的肽命中率有关。
