Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.
Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.
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DOI：
10.1021/acs.analchem.1c04200
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发表时间：
2022-07-26
影响因子：
7.4
通讯作者：
Samant, Rahul S.
中科院分区：
文献类型：
--
作者：
Johnston, Harvey E.;Yadav, Kranthikumar;Kirkpatrick, Joanna M.;Biggs, George S.;Oxley, David;Kramer, Holger B.;Samant, Rahul S.
完整的，可重复的蛋白质物质提取是必要的全面和公正的蛋白质组分析。目前的金标准是单锅固相增强样品制备（SP3），其中有机溶剂和磁珠用于变性和捕获蛋白质聚集体，随后洗涤去除污染物。然而，SP3依赖于有效的蛋白质固定在珠上，在洗涤步骤中有损失的风险，并且在更高的蛋白质投入下表现出损失和更高的成本。在这里，我们提出溶剂沉淀SP3 （SP4）作为SP3蛋白清理的替代方法，通过短暂离心而不是磁力捕获乙腈诱导的蛋白聚集体，使用可选的低成本惰性玻璃珠来简化处理。SP4在1-5000 μg制剂中恢复了相同或更高的蛋白质产量，并提高了重复性(中位蛋白质R2 0.99 （SP4) vs 0.97 (SP3)）。深层蛋白质组学分析表明，SP4比SP3更能回收低溶解度和跨膜蛋白质，有利于在80%和50%的有机溶剂下聚集蛋白质，并且SP4和S-Trap的回收率相当。SP4在其他三个实验室中通过八种样品类型和五种裂解缓冲液进行了验证-所有这些都证实了与SP3相同或改进的蛋白质组特性。在几乎相同的回收率下，这项工作进一步说明了蛋白质沉淀是SP3蛋白清理的主要机制，并确定了磁捕获有损失的风险，特别是在更高的蛋白质浓度和更疏水的蛋白质中。SP4提供了一种极简的蛋白质清理方法，提供了成本效益高的输入可扩展性，完全省略珠子的选项，并为SP3应用程序提出了重要的考虑因素，同时保留了SP3的速度和兼容性。
Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism—with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1–5000 μg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers—all confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications—all while retaining the speed and compatibility of SP3.
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影响因子：
4.1
作者：
Jiang, L;He, L;Fountoulakis, M
通讯作者：
Fountoulakis, M
影响因子：
7.4
作者：
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通讯作者：
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DOI：
10.1074/mcp.ra120.002411
发表时间：
2021
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
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通讯作者：
Wohlschlegel JA
DOI：
10.1002/rcm.8183
发表时间：
2018-08-30
期刊：
Rapid communications in mass spectrometry : RCM
影响因子：
--
作者：
Kay RG;Challis BG;Casey RT;Roberts GP;Meek CL;Reimann F;Gribble FM
通讯作者：
Gribble FM
影响因子：
9.9
作者：
Leutert, Mario;Rodriguez-Mias, Ricard A.;Villen, Judit
通讯作者：
Villen, Judit