Fractionation of peptides in proteomics with the use of pI-based approach and ZipTip pipette tips
Fractionation of peptides in proteomics with the use of pI-based approach and ZipTip pipette tips
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DOI：
10.1016/j.jpba.2003.11.001
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发表时间：
2004-03-10
影响因子：
3.4
通讯作者：
Baczek, T
中科院分区：
文献类型：
--
作者：
Baczek, T
这项工作的目的是探索溶液中等电聚焦(Sef)分馏方法的实用性。该方法被证明是蛋白质组学中蛋白质胰酶混合物的替代分离方法。利用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF/TOF-MS)对多肽进行鉴定。在该研究中，以前设计了基于聚丙烯酰胺的小型化多室分馏装置(每室75个体积)，使用了凝胶膜和固定化技术。为了评价分离分离结合MS/MS鉴定多肽的效率和准确性，采用牛血清白蛋白(BSA)消化和五种蛋白质的混合消化。对这种简单的多肽混合物进行的研究证明，Sef设备能够将多肽主要集中在一个腔室中。此外，对识别的多肽进行了pI(Calc)和pI(Exp)之间的相关性分析，证明了获得实验上有用的高相关性的可能性。这一信息被发现在鉴定的蛋白质之间的假阳性评估过程中具有潜在的构建额外约束的价值。接下来，将Sef分级的研究与ZipTip移液器针尖在简单蛋白质混合情况下对分级多肽的实际使用进行了评估。为此，使用了五个蛋白质消化样本。这种分析没有事先进行任何分级，因此能够鉴定出数量非常有限的蛋白质。在MS分析前，单独使用Sef或与ZipTips分离联合使用可获得显著的改善。基于溶液等电聚焦的分离方法是一种高效、准确的蛋白质酶切分离方法，可作为蛋白质组学二维分离的第一个有用维度。此外，来自基于PI的分馏方法的分析信息可以被视为数据库匹配约束的附加来源。它也可以成为蛋白质组学中肽分离的分析和生物信息学研究的有价值的工具。(C)2003爱思唯尔B.V.保留所有权利。
The aim of the work was to explore the utility of the in-solution isoelectric focusing (sIEF) fractionation method. That method was proved to be the alternative separation method of mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). For that research, previously designed the miniaturized multi-chamber fractionation sIEF device (75 mul volume for each chamber) based on polyacrylamide, gel membranes with immobilines technology was utilized. To evaluate the efficiency and accuracy of OFF fractionation combined with MS/MS peptides identification, bovine serum albumin (BSA) digest and mixture of five proteins digest were used.First, fractionation of bovine serum albumin digest sample was performed using sIEF method. Studies performed for that simple mixture of peptides proved the ability of the sIEF device to focus peptides mostly in one chamber. Additionally performed the correlation analysis between pI(calc) and pI(exp) values for identified peptides proved the possibility to obtain experimentally useful high correlation. That information was found to have a potential value for construction of additional constraint during false positives evaluation process among identified proteins. Next, studies on the sIEF fractionation were combined with the evaluation of practical use of ZipTip pipette tips to fractionate peptides in the case of simple mixture of proteins. For this, five proteins digest samples were used. The analysis without prior any fractionation enabled to identify very limited number of proteins. The significant improvement was obtained when one used sIEF alone or with combination with ZipTips fractionation prior to MS analysis. The proposed approach based on in-solution isoelectric focusing proved to be an efficient and accurate alternative fractionation method of protein digests and can be considered as the first useful dimension in two-dimensional proteomics separations. Moreover, analytical information from that pI-based fractionation method can be considered as the additional source of database matching constraint. It can also be a valuable tool for analytical and bioinformatic studies of peptides fractionation in proteomics. (C) 2003 Elsevier B.V. All rights reserved.