Boschetti Egisto, Righetti Pier Giorgio
BioRad Laboratories, C/o CEA-Saclay, Gif-sur-Yvette, France.
Proteomics. 2009 Mar;9(6):1492-510. doi: 10.1002/pmic.200800389.
The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low-abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the "deep proteome", such as: depletion of high-abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1-pH-unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off-Gel electrophoresis. The physico-chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the "minority" proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminating r-DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg-white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho- and glyco-proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off-Gels, and the like.
本综述涉及由六肽组成的组合配体文库在低丰度蛋白质组捕获和浓缩中的应用。该方法于2005年首次报道，与目前旨在探索“深度蛋白质组”的其他方法进行了比较，例如：通过单独或联合抗体（针对最丰富物种的多达20种抗体）去除高丰度蛋白质（特别是在血清和脑脊液中）；窄（1个pH单位）IPG（变焦凝胶）以及使用多室电解槽或离线凝胶电泳进行预分级分离。还探索了六肽文库的物理化学性质，即评估诱饵的合适长度以及较短寡肽的行为，直至单个氨基酸引发的捕获。给出了该文库使用的一些示例，如生物体液（人血清、尿液、胆汁、脑脊液）和细胞裂解物（血小板、红细胞）的分析。在所有情况下，与未处理的对照样品相比，检测到的蛋白质数量是其3至5倍。也许最引人注目的结果是在红细胞蛋白质组中获得的，在“少数”蛋白质组中可以鉴定出1570种蛋白质，仅占总细胞裂解物的2%。另一个有趣的应用领域涉及浓缩和检测污染供人类食用的重组DNA蛋白质的痕量杂质：首次发现了几种以前从未报道过的宿主蛋白质。目前正在研究其他非人类样品，如蛋清（可鉴定出不少于148种独特基因产物）、蛋黄（有255种独特物种）和巴西橡胶树的乳胶。预计该配体文库可能是检测不同病理和药物治疗生物标志物的非常有用的工具。展望未来，可以设想合成能够捕获特定类蛋白质（如磷酸化和糖基化蛋白质）的专用文库。此外，该文库可与目前流行的其他技术（如变焦凝胶、离线凝胶等）联合使用。
