Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, CA , USA ; Department of Bioengineering, University of California Berkeley , Berkeley, CA , USA.
Front Bioeng Biotechnol. 2015 Apr 1;3:44. doi: 10.3389/fbioe.2015.00044. eCollection 2015.
Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Key to the success of these efforts has been transformational developments in feedstock characterization and metabolic engineering of biofuel-producing microbes. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utilization of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. Nano-flow liquid chromatography coupled to high-mass accuracy, high-resolution mass spectrometers has become the dominant approach for the analysis of complex proteomic samples, yet such assays still require dedicated experts for data acquisition, analysis, and instrument upkeep. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased throughput of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chromatography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms commonly used as model systems for lignocellulosic biofuels research. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of complex samples. To the best of our knowledge, this study represents the first attempt to validate the standard flow approach for shotgun proteomics.
在过去的 10 年中，生物能源领域取得了重大成就，这鼓励了许多后续努力，这些努力主要集中在合成生产类似燃料的化合物上。这些努力的关键是在生物燃料生产微生物的原料特性和代谢工程方面的变革性发展。远远落后于这些进展的是用于生物能源领域感兴趣的系统进行特征描述和量化的分析方法。特别是，蛋白质组学的应用虽然对于鉴定新的酶和诊断与生物燃料生产微生物相关的问题很有价值，但由于缺乏稳健性和有限的通量而受到限制。纳米流液相色谱与高质量、高分辨率质谱仪的结合已成为分析复杂蛋白质组学样品的主要方法，但此类测定仍然需要专门的专家进行数据采集、分析和仪器维护。最近采用标准流量色谱（约 0.5mL/min）进行靶向蛋白质组学，突出了该方法在样品分析中的稳健性和增加的通量。因此，我们评估了标准流量液相色谱在使用大肠杆菌和拟南芥样品进行 shotgun 蛋白质组学中的适用性，大肠杆菌和拟南芥通常被用作木质纤维素生物燃料研究的模型系统。使用标准流量色谱进行 120 分钟梯度实验，我们能够从大肠杆菌样品中常规鉴定出近 800 种蛋白质；而对于拟南芥的样品，可以可靠地鉴定出 1000 多种蛋白质。对鉴定出的肽的分析表明，该方法适用于 shotgun 蛋白质组学的可重复应用。标准流量液相色谱在 shotgun 蛋白质组学中提供了一种用于分析复杂样品的稳健方法。据我们所知，这项研究是首次尝试验证 shotgun 蛋白质组学的标准流量方法。
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