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A Streamlined Sample Preparation Method for Mass Spectrometric Analysis

机译：质谱分析的流线型样品制备方法

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摘要

Mass spectrometry-based proteomic technology experienced remarkable ad-vancement in the past decades. However, their application was still hamperedby the complexity of sample preparation. Conventional strategies for samplepreparation incorporate multiple time-consuming steps, including cell lysis,protein extraction, protease cleavage, and desalting. Thus, we explored a sim-plied method (the cell-absorb method) during which living cells were absorbedinto vacuum-dried polyacrylamide gel and directly digested in gel into peptidesfor subsequent LC-MS/MS analysis. As a consequence, both of the steps forcell lysis and protein extraction involved in traditional protocol were skipped.In addition to the decrease in time, more proteins were identied. Indeed,3022proteins were identied by the cell-absorb method. Meanwhile, only 2642 and2420 proteins were identied by the classical SDS-PAGE based method andthe reported gel absorption-based method, respectively. The cell-absorb methodexhibited apparent advantagein terms of the depth of proteome coverage. Fur-thermore, the number of proteins identied show excellent reproducibility witha CV (coefcient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promis-ing choice for mapping the whole proteome of cells.
机译：基于质谱的蛋白质组学技术在过去的几十年中经历了显着的宣传风险。然而，他们的应用仍然是妨碍样品制备的复杂性。样品制备的常规策略包含多个耗时的步骤，包括细胞裂解，蛋白质提取，蛋白酶切割和脱盐。因此，我们探讨了一种模拟方法（细胞 - 吸收方法），在此期间活细胞被吸收细胞被吸收真空干燥的聚丙烯酰胺凝胶，并在凝胶中直接消化为随后的LC-MS / MS分析。因此，跳过了对传统方案中涉及的裂解裂解和蛋白质提取的步骤。除了减少时，鉴定了更多的蛋白质。实际上，细胞吸收方法鉴定了3022颗粒。同时，仅通过基于古典SDS-PAGE的方法和报告的凝胶吸收的方法鉴定了2642和2420蛋白。细胞吸收方法抑制蛋白质组覆盖深度的表观优势。毛虫，所鉴定的蛋白质的数量表现出使用细胞 - 吸收方法的三种重复的CV（变异系数）的优异再现性（变异的细胞）。这些优势表明，细胞吸收方法是用于映射细胞的整个蛋白质组的促销选择。

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