Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.
Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.
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胰蛋白酶经常被用来在质谱鉴定之前切割蛋白质。传统上，酶消化涉及在37°C下对蛋白质进行过夜孵育，这是耗时的，尽管仍然可能产生较低的消化效率。虽然提高温度理论上会加速消化，但它也会破坏酶的稳定，促进胰蛋白酶的失活。因此，我们质疑温度升高是否有利于改善胰蛋白酶消化。在这里，我们量化了钙稳定胰蛋白酶在高温下的蛋白质消化动力学，并强调钙离子对保存酶的关键重要性。我们定量地证明，与传统的（隔夜，37°C）蛋白质组处理相比，在47°C下处理1小时可以提供更好的消化。我们的增强消化方案的实际影响是通过自下而上的复杂蛋白质组混合物的质谱分析显示。自下而上的蛋白质组学依赖于高效的胰蛋白酶消化在质谱分析之前。先前的研究表明，在高温下消化可以加速蛋白质水解，显示出ms鉴定的肽的数量增加。然而，改善的序列覆盖可能是部分消化的结果，因为较高的温度使酶不稳定并降解，导致增强的活性是短暂的。在这里，我们使用光谱（BAEE）测定在消化的整个时间过程中定量钙稳定胰蛋白酶活性。在47°C时，钙的加入使胰蛋白酶的稳定性提高了25倍。较高的温度表明累积胰蛋白酶活性的净下降。通过对酵母蛋白质组提取物进行自下而上的质谱分析，我们证明在47°C和10 mM Ca2+条件下消化1小时可使肽鉴定总数增加29%。同时，在47°C的酶切过程中，缺失1个或更多切割位点的肽的定量比例减少，支持1 h方案的酶切效率提高。胰蛋白酶特异性也得到改善，如半胰蛋白酶肽的定量丰度下降所见。我们的增强消化方案提高了自下而上样品制备的吞吐量，并验证了该方法是一种强大的、低成本的替代方案，可以最大限度地提高蛋白质消化效率。
Trypsin is frequently employed to cleave proteins ahead of mass spectrometry characterization. Traditionally, enzyme digestion involves overnight incubation of proteins at 37 °C, which is time consuming though still may yield poor digestion efficiency. While raising the temperature should theoretically accelerate the digestion, it also destabilizes the enzyme and promotes trypsin de-activation. We therefore questioned whether elevated temperature is beneficial for improving tryptic digestion. Here, we quantify protein digestion kinetics at elevated temperatures for calcium-stabilized trypsin and enforce the critical importance of calcium ions to preserve the enzyme. We quantitatively demonstrate that 1 h at 47 °C provides a superior digest when compared to conventional (overnight, 37 °C) processing of the proteome. The practical impact of our enhanced digestion protocol is shown through bottom-up mass spectrometry analysis of a complex proteome mixture. Bottom-up proteomics relies on efficient trypsin digestion ahead of MS analysis. Prior studies have suggested digestion at elevated temperature to accelerate proteolysis, showing an increase in the number of MS-identified peptides. However, improved sequence coverage may be a consequence of partial digestion, as higher temperatures destabilize and degrade the enzyme, causing enhanced activity to be short-lived. Here, we use a spectroscopic (BAEE) assay to quantify calcium-stabilized trypsin activity over the complete time course of a digestion. At 47 °C, the addition of calcium contributes a 25-fold enhancement in trypsin stability. Higher temperatures show a net decrease in cumulative trypsin activity. Through bottom-up MS analysis of a yeast proteome extract, we demonstrate that a 1 h digestion at 47 °C with 10 mM Ca2+ provides a 29% increase in the total number of peptide identifications. Simultaneously, the quantitative proportion of peptides with 1 or more missed cleavage sites was diminished in the 47 °C digestion, supporting enhanced digestion efficiency with the 1 h protocol. Trypsin specificity also improves, as seen by a drop in the quantitative abundance of semi-tryptic peptides. Our enhanced digestion protocol improves throughput for bottom-up sample preparation and validates the approach as a robust, low-cost alternative to maximized protein digestion efficiency.
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影响因子：
4.1
作者：
Daniel, Roy M.;Peterson, Michelle E.;Lee, Charles K.
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Lee, Charles K.
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2
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Jeng, Jingyueh;Lin, Mon-Fang;Shiea, Jentaie
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Shiea, Jentaie
影响因子：
7.4
作者：
Havlis, J;Thomas, H;Shevchenko, A
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Shevchenko, A
影响因子：
4.4
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Cannon J;Lohnes K;Wynne C;Wang Y;Edwards N;Fenselau C
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Fenselau C
影响因子：
4.4
作者：
Hains, Peter G.;Robinson, Phillip J.
通讯作者：
Robinson, Phillip J.