Standard flow liquid chromatography for shotgun proteomics in bioenergy research.
Standard flow liquid chromatography for shotgun proteomics in bioenergy research.
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DOI：
10.3389/fbioe.2015.00044
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发表时间：
2015
影响因子：
5.7
通讯作者：
Petzold CJ
中科院分区：
文献类型：
--
作者：
González Fernández-Niño SM;Smith-Moritz AM;Chan LJ;Adams PD;Heazlewood JL;Petzold CJ
在过去的10年里，生物能源领域取得了重大成就，这鼓励了许多人继续努力，以生物合成生产类似燃料的化合物。这些努力取得成功的关键是原料表征和生物燃料生产微生物代谢工程的转型发展。远远落后于这些进步的是分析方法来表征和量化生物能源领域感兴趣的系统。特别是，蛋白质组学的利用，虽然对鉴定新的酶和诊断与生物燃料生产微生物相关的问题有价值，但由于缺乏鲁棒性和有限的吞吐量而受到限制。纳米流液相色谱与高质量精度、高分辨率质谱仪相结合，已成为分析复杂蛋白质组学样品的主要方法，但这种分析仍然需要专门的专家进行数据采集、分析和仪器维护。最近采用标准流动色谱法（约0.5 mL/min）进行靶向蛋白质组学分析，强调了这种方法的稳定期和提高了样品分析的吞吐量。因此，我们利用大肠杆菌和拟南芥这两种通常用作木质纤维素生物燃料研究模型系统的生物样本，评估了标准流动液相色谱法在霰弹枪蛋白质组学中的适用性。采用标准流式色谱120分钟梯度，我们能够从大肠杆菌样品中常规鉴定近800种蛋白质；而对于拟南芥的样本，可以可靠地识别出超过1000种蛋白质。对鉴定的多肽的检测表明，该方法适用于鸟枪蛋白质组学的可重复性应用。散弹枪蛋白质组学的标准流动液相色谱法为复杂样品的分析提供了一种可靠的方法。据我们所知，这项研究代表了验证shotgun蛋白质组学标准流程方法的第一次尝试。
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.
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影响因子：
3.7
作者：
Cetinkol ÖP;Smith-Moritz AM;Cheng G;Lao J;George A;Hong K;Henry R;Simmons BA;Heazlewood JL;Holmes BM
通讯作者：
Holmes BM
影响因子：
3.4
作者：
Asara, John M.;Christofk, Heather R.;Cantley, Lewis C.
通讯作者：
Cantley, Lewis C.
影响因子：
32.5
作者：
DeMartini, Jaclyn D.;Pattathil, Sivakumar;Wyman, Charles E.
通讯作者：
Wyman, Charles E.
DOI：
10.1016/j.jasms.2009.03.007
发表时间：
2009-07-01
影响因子：
3.2
作者：
Gapeev, Alexei;Berton, Alberto;Fabris, Daniele
通讯作者：
Fabris, Daniele
DOI：
10.1081/jlc-200053046
发表时间：
2005-01-01
影响因子：
1.3
作者：
Swartz, ME
通讯作者：
Swartz, ME