中文摘要：

【目的】提高蛋白质胶内酶解效率，减少萃取过程中肽段的损失，增加质谱鉴定的成功率。【方法】在常规胶内酶解方法的基础上，优化脱色、酶解、萃取等步骤，包括：用铁氰化钾和硫代硫酸钠对银染胶块脱色，酶解温度提高至55％，以40mmol／L碳酸氢铵作为酶切缓冲液，使用50％乙腈（ACN）水溶液3步萃取；并使用一步法胶内酶解探索快速胶内酶解方法。【结果】使用铁氰化钾和硫代硫酸钠可在短时间内将银染胶块完全脱色，55℃温浴可使酶切时问由20h减少为2h，以40mmol／I碳酸氢铵作为酶切缓冲液，可省去ZipTip脱盐步骤。优化后，质谱鉴定率提高30．0％（绝对值），且能明显高酶切效率，使目标肽段更高丰度地呈现出来。【结论】优化后的胶内酶解法可显著提高蛋白质胶内酶解的质谱鉴定成功率和肽段覆盖率，其中一步法胶内酶解法有效减少了胶内酶解时间和步骤，可用于高丰度蛋白质的快速质谱检测。

英文摘要：

[Objective]The studies were carried out in order to increase the efficiency of protein in-gel digestion, reduce the loss of peptides extraction and to increase the ratio of mass spectrometry identification. [Method]The procedures of decoloring, digestion and extraction were optimized using following methods： （i）ferricyanide and sodium thiosulfate reagents were used for decoloring of solution, （ii） for enzyme digestion, 40 mmol/L ammonium bicarbonate buffer was used at 55℃ temperature, （iii） three steps extraction was performed with 50% acetonitrile （ACN） and （iv） established one-step in-gel digestion method. [Result]Compared to the control, use of ferricyanide and sodium thiosulfate reagents decolored the gel pieces quickly and completely. The enzyme digestion time reduced to 2 h from 20 h at 55% temperature, and the Zip- Tip desahing procedure was replaced with 40 mmol/L ammonium bicarbonate as enzmye digestion buffer. The ratio of mass spectrometry was increased by 30% （absolute value）, the efficiency of enzyme digestion was also enhanced which resulted in highly abundant peptides. [Conclusion]The optimized method significantly improved the ratio of mass spectrometry identification and peptides coverage. One-step method significantly reduced the time of in-gel digestion and facilitated fast identification of highly abundant proteins.