Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics
Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics
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DOI：
10.1021/pr300192f
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发表时间：
2012-05-01
影响因子：
4.4
通讯作者：
Roepstorff, Peter
中科院分区：
文献类型：
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作者：
Nogueira, Fabio C. S.;Palmisano, Giuseppe;Roepstorff, Peter
在蛋白质组学研究中，质谱学已经成为多肽和蛋白质定量所不可缺少的。当蛋白质组学技术被应用于了解植物生物学时，双向凝胶电泳法仍然是蛋白质分离、鉴定和定量的流行方法。在本工作中，我们使用LC-MS比较了同位素(ICPL)和等压(ITRAQ)化学标记技术来定量测定蓖麻种子胚乳中三个发育阶段(IV、VI和X)的蛋白质。每一阶段的胚乳蛋白在溶液中经胰酶消化后，用ICPL和iTRAQ两个顺序(正向和反向)标记等量的多肽。每个样品都被提交给与LTQ-Orbitrap高分辨率质谱仪耦合的NanoLC。比较标记性能，iTRAQ能够标记99.8%的唯一多肽，而ICPL标记94.1%。经过统计分析，可以量化309(ICPL)和321(ITRAQ)蛋白，其中95个是ICPL特异的，107个是iTRAQ特异的，214个是两种标记策略共同的。我们注意到iTRAQ的量化可能会受到标签的影响。尽管iTRAQ和ICPL在蛋白质定量方面的效率取决于几个参数，但这两种标记方法都能够成功地定量蓖麻种子发育过程中胚乳中存在的蛋白质，并结合使用，增加了定量蛋白质的数量。
Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.