Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.
Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.
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DOI：
10.1186/1477-5956-9-24
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发表时间：
2011-05-09
期刊：
影响因子：
2
通讯作者：
Wang ZL
中科院分区：
文献类型：
--
作者：
Liu B;Qiu FH;Voss C;Xu Y;Zhao MZ;Wu YX;Nie J;Wang ZL
高丰度蛋白质的去除是血清/血浆蛋白质组学研究中的一个主要挑战。在本研究之前，大多数用于去除高丰度蛋白质的市售试剂盒仅在成人血清/血浆中进行了检测和评价，而脐带血清/血浆的去除效率尚未阐明。一些成人和胎儿蛋白质（如白蛋白）之间的结构差异使得成人和脐带血清/血浆的消除方法可能是可变的。因此，本研究的主要目的是研究几种常用的商业试剂盒在从脐带血清中去除高丰度蛋白质时的效率，并确定哪种试剂盒产生最有效和可重复的结果，用于进一步的脐带血清蛋白质组学研究。在脐带血清样本中，基于免疫亲和性的试剂盒（PROTIA-Sigma和5185-Agilent）显示出比基于固定化染料的试剂盒（PROTBA-Sigma）更高的消除效率。PROTIA-Sigma和5185-Agilent试剂盒在连续使用三次时均保持了较高的去除效率。通过PROTIA-Sigma试剂盒的消耗，通过减少由高丰度蛋白质的存在产生的模糊条带和增加其他蛋白质斑点的强度，改善了2DE凝胶质量。在使用相同检测参数的图像分析期间，在粗血清凝胶中检测到411 ± 18个斑点，而在耗尽血清凝胶中检测到757 ± 43个斑点。通过MALDI-TOF/TOF MS鉴定了耗尽血清凝胶特有的8个斑点，其中7个是低丰度蛋白质。基于免疫亲和的试剂盒在脐带血清样品的高丰度蛋白质去除方面超过了基于固定化染料的试剂盒，并且显著提高了用于检测痕量生物标志物的2DE凝胶质量。
High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.
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