Miniprep assisted proteomics (MAP) for rapid proteomics sample preparation.
Miniprep assisted proteomics (MAP) for rapid proteomics sample preparation.
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DOI：
10.1039/d2ay01549h
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发表时间：
2023-02-16
影响因子：
3.1
通讯作者：
Champion, Matthew M. M.
中科院分区：
文献类型：
--
作者：
Mousseau, C. Bruce;Pierre, Camille A. A.;Hu, Daniel D. D.;Champion, Matthew M. M.
通过使用去污剂进行变性和增溶，大大改善了自下而上蛋白质组学中蛋白质的完全酶促消化。然而，去污剂与许多蛋白酶不相容并且对LC-MS/MS高度有害。最近，基于过滤器的方法由于其在消化和质谱分析之前去除去污剂和有害试剂的能力而得到广泛使用。我们假设，非特异性蛋白质结合带负电荷的硅基过滤器将增强通过添加感胶盐，类似于DNA纯化。我们试图利用这些相互作用，并研究低成本的DNA纯化旋转过滤器，“Minipreps”，是否有效地和可重复地结合蛋白质进行消化和LC-MS/MS分析。我们提出了一种新的样品制备方法--微量制备辅助蛋白质组学（MAP）。我们证明了使用MAP的蛋白质和全细胞裂解物的结合能力，性能，回收率和识别率。以商业捕获制剂为基准，MAP从0.5-50 μg分析中回收了等同或更高的蛋白质产率。大肠杆菌裂解物的纳米UHPLC-MS/MS蛋白质组分析与现有方法相比具有99.3%的重叠，并且在1%FDR蛋白质水平下重复微量制备物的再现性为98.8%。进行了无标记定量蛋白质组学，91.2%的定量蛋白质的%CV <20%（2044/2241）。微量制备辅助蛋白质组学可以在几分钟内完成，显示低变异性，高回收率和蛋白质组深度。这表明在开发新的蛋白质组学样品制备技术中偶然结合的重要作用。MAP是一种高效、超低成本的样品制备替代品，可在商业上获得，每小量制备成本为0.50美元。微量制备辅助蛋白质组学（MAP）是通过与Si-DNA微量制备物的偶然结合来进行自下而上的蛋白质组学样品制备的快速方法。这结合了商业生产的柱的一致性和内部设备的低成本。
Complete enzymatic digestion of proteins for bottom-up proteomics is substantially improved by use of detergents for denaturation and solubilization. Detergents however, are incompatible with many proteases and highly detrimental to LC-MS/MS. Recently; filter-based methods have seen wide use due to their capacity to remove detergents and harmful reagents prior to digestion and mass spectrometric analysis. We hypothesized that non-specific protein binding to negatively charged silica-based filters would be enhanced by addition of lyotropic salts, similar to DNA purification. We sought to exploit these interactions and investigate if low-cost DNA purification spin-filters, ‘Minipreps,’ efficiently and reproducibly bind proteins for digestion and LC-MS/MS analysis. We propose a new method, Miniprep Assisted Proteomics (MAP), for sample preparation. We demonstrate binding capacity, performance, recovery and identification rates for proteins and whole-cell lysates using MAP. MAP recovered equivalent or greater protein yields from 0.5–50 μg analyses benchmarked against commercial trapping preparations. Nano UHPLC-MS/MS proteome profiling of lysates of Escherichia coli had 99.3% overlap vs. existing approaches and reproducibility of replicate minipreps was 98.8% at the 1% FDR protein level. Label Free Quantitative proteomics was performed and 91.2% of quantified proteins had a %CV <20% (2044/2241). Miniprep Assisted Proteomics can be performed in minutes, shows low variability, high recovery and proteome depth. This suggests a significant role for adventitious binding in developing new proteomics sample preparation techniques. MAP represents an efficient, ultra-low-cost alternative for sample preparation in a commercially obtainable device that costs ∼$0.50 (USD) per miniprep. Miniprep Assisted Proteomics (MAP) is a rapid approach to bottom-up proteomics sample preparation by adventitious binding to Si-DNA minipreps. This combines the consistency of a commercially produced column with the low-cost of in-house devices.
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影响因子：
3.6
作者：
Champion, MM;Campbell, CS;Hu, JC
通讯作者：
Hu, JC
影响因子：
7.4
作者：
Johnston, Harvey E.;Yadav, Kranthikumar;Kirkpatrick, Joanna M.;Biggs, George S.;Oxley, David;Kramer, Holger B.;Samant, Rahul S.
通讯作者：
Samant, Rahul S.
影响因子：
4.4
作者：
Erde, Jonathan;Loo, Rachel R. Ogorzalek;Loo, Joseph A.
通讯作者：
Loo, Joseph A.
DOI：
10.1146/annurev-anchem-071015-041535
发表时间：
2016-01-01
期刊：
ANNUAL REVIEW OF ANALYTICAL CHEMISTRY, VOL 9
影响因子：
--
作者：
Gillet, Ludovic C.;Leitner, Alexander;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
4.4
作者：
Elinger, Dalia;Gabashvili, Alexandra;Levin, Yishai
通讯作者：
Levin, Yishai