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首页> 外文期刊>Proteome science >Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae
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Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

机译：酿酒酵母中胞浆未折叠蛋白反应的二维凝胶电泳实验中一维IPG和NEPHGE技术的比较

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Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI?>?7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load. Conclusions Comparison of broad range (pH 3–10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH 4–7) IPG technique is a method of choice for the analysis of acidic proteins.
机译：背景技术二维凝胶电泳（2DE）是蛋白质组学中最流行的方法之一。目前，大多数2DE实验都是在第一维使用固定的pH梯度（IPG）进行的;然而，一些实验室仍使用基于载流子两性电解质的等电聚焦技术。这项研究的目的是通过使用相同的样品和相同的二维方法，直接比较基于IPG和基于非平衡pH梯度电泳（NEPHGE）的2DE技术。我们已经分别使用了用于IPG和NEPHGE的市售Invitrogen ZOOM IPGRunner和WITAvision系统。通过分析酿酒酵母中胞质未折叠蛋白反应（UPR-Cyto）过程中差异蛋白的表达，比较了基于IPG和基于NEPHGE的2DE方法的有效性。结果在基于IPG的方法中2DE程序中的蛋白质损失更高，尤其是对于碱性（pI？>？7）蛋白质而言。基于NEPHGE的方法中斑点的总体重现性稍好;但是，在评估碱性和酸性蛋白质斑点时存在明显差异。使用考马斯染色，大约一半的检测到的碱性蛋白斑点不能被基于IPG的2DE重现，而基于NEPHGE的方法在碱性凝胶区显示出极好的重现性。两种方法中酸性蛋白的重现性相似。在两种2DE技术中，单独蛋白质斑点的绝对和相对体积变异性均相当。关于UPR-Cyto的蛋白质组学分析，结果举例说明了该方法的一般比较参数。通过基于NEPHGE的2DE方法鉴定了在UPR-Cyto胁迫期间过表达的新的高度碱性蛋白Sis1p，而基于IPG的方法在基本pI范围内显示了不可靠的结果，并且未提供有关基本UPR-Cyto蛋白的任何新信息。在酸性范围内，两种方法均检测并定量了主要的UPR-Cyto蛋白。基于NEPHGE的2DE方法的缺点是无法检测某些高酸性蛋白质。 NEPHGE的优点是蛋白质容量更高，在高蛋白质负载下具有良好的重现性和斑点质量。结论比较宽范围（pH 3-10）的基于2DE的方法表明，基于NEPHGE的方法在分析碱性蛋白质方面优于IPG（Invitrogen）的2DE方法。不过，窄范围（pH 4-7）IPG技术是分析酸性蛋白质的一种选择方法。

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