Comprehensive Comparison of iTRAQ and Label-free LC-Based Quantitative Proteomics Approaches Using Two Chlamydomonas reinhardtii Strains of Interest for Biofuels Engineering
Comprehensive Comparison of iTRAQ and Label-free LC-Based Quantitative Proteomics Approaches Using Two Chlamydomonas reinhardtii Strains of Interest for Biofuels Engineering
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DOI：
10.1021/pr2008225
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发表时间：
2012-01-01
影响因子：
4.4
通讯作者：
Hicks, Leslie M.
中科院分区：
文献类型：
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作者：
Wang, Hongxia;Alvarez, Sophie;Hicks, Leslie M.
定量蛋白质组学技术的全面比较在文献中是罕见的，但他们是至关重要的最佳选择的方法和方法，是理想的一个给定的蛋白质组学计划。在这项研究中，两种基于LC的定量蛋白质组学方法-iTRAQ和无标记-使用LTQ-Orbitrap Velos平台实现。为了进行比较，使用的模型是在替代生物燃料生产的背景下两种莱茵衣藻菌株的总蛋白质含量。菌株比较包括sta 6（cw 15的无淀粉突变体），其产生的含有三酰甘油（TAG）的脂质体（LB）是其亲本菌株cw 15（细胞壁缺陷型C. reinhardtii菌株）。使用内标物添加严格评估每种方法的定量准确度和精密度。将使用HCD（高能碰撞诱导解离）片段化的iTRAQ 4plex标记结果与使用基于完整肽峰面积和碰撞诱导解离的无标记方法获得的结果进行比较。对这两种定量蛋白质组学方法的准确度和精密度、鉴定/定量蛋白质的数量、检测到的统计学显著性蛋白质差异以及效率进行了评价和比较。对每种菌株进行四次技术重复和三次生物重复，以评估两种方法的技术和生物变异。共鉴定出896和639种蛋白质，具有高置信度，使用生物重复，分别用无标记和iTRAQ对329和124种蛋白质进行了显著定量。结果表明，iTRAQ标记和无标记方法都提供了高质量的定量和定性数据，使用nano-LC耦合LTQ：Orbitrap Velos质谱仪，但最佳方法的选择取决于实验设计和要解决的生物学问题。cw 15和sta 6之间的差异蛋白的功能分类揭示了已知的但也是可能负责在sta 6中产生脂质的新机制，并为旨在工程改造这些菌株以获得高油产量的未来研究设定了基线。
Comprehensive comparisons of quantitative proteomics techniques are rare in the literature, yet they are crucially important for optimal selection of approaches and methodologies that are ideal for a given proteomics initiative. In this study, two LC-based quantitative proteomics approaches-iTRAQ and label-free-were implemented using the LTQ-Orbitrap Velos platform. For this comparison, the model used was the total protein content from two Chlamydomonas reinhardtii strains in the context of alternative biofuels production. The strain comparison includes sta6 (a starch-less mutant of cw15) that produces twice as many lipid bodies (LB) containing triacylglycerols (TAGs) as its parental strain cw15 (a cell wall-deficient C. reinhardtii strain) under nitrogen starvation. Internal standard addition was used to rigorously assess the quantitation accuracy and precision of each method. Results from iTRAQ4plex labeling using HCD (higher energy collision-induced dissociation) fragmentation were compared to those obtained using a label-free approach based on the peak area of intact peptides and collision-induced dissociation. The accuracy and precision, number of identified/quantified proteins and statistically significant protein differences detected, as well as efficiency of these two quantitative proteomics methods were evaluated and compared. Four technical and three biological replicates of each strain were performed to assess both the technical and biological variation of both approaches. A total of 896 and 639 proteins were identified with high confidence, and 329 and 124 proteins were quantified significantly with label-free and iTRAQ respectively, using biological replicates. The results showed that both iTRAQ labeling and label-free methods provide high quality quantitative and qualitative data using nano-LC coupled with the LTQ:Orbitrap Velos mass spectrometer, but the selection of the optimal approach is dependent on experimental design and the biological question to be addressed. The functional categorization of the differential proteins between cw15 and sta6 reveals already known but also new mechanisms likely responsible for the production of lipids in sta6 and sets the baseline for future studies aimed at engineering these strains for high oil production.