摘要
目的:改善胶内酶切的肽段回收率,提高蛋白质的有效鉴定率。方法:将改进的胶内酶切“四步提取法”,应用到96个双向电泳蛋白质斑点分析。结果:该方法提高了胶内蛋白质酶切肽段的回收率,改善了质谱图谱的质量。蛋白质的有效鉴定率达89.6%,其中,数据库检索得分80分以上者达82%。另外,证实了酶解原液直接用于质谱分析可获得较好信噪比的谱图。结论:应用“四步提取法”使得酶解肽段提取率和蛋白质的有效鉴定率明显提高。
Objective: To improve the peptide recovery during in-gel digestion. Methods and Results: Details of the 'four-step extraction' of in-gel digestion were discussed in this paper. 96 of the protein spots from two dimensional gel electrophoresis were processed for evaluating this protocol. Results showed that this protocol enhanced the peptide recovery during in-gel digestion and improved the quality of MALDI-TOF-MS peptide mass fingerprinting (PMF). About 89.6% of protein spots were identified and 82% of the identified proteins obtained the scores of higher than 80 by MASCOT search engine. Also, direct PMF analysis of the digestion resulting solution was studied. Conclusion: The application of ' four-step extraction' method enhances the peptide recovery during in-gel digestion,and increases identification of the protein.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第2期168-172,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家"863"计划发展项目(2001AA233041)
关键词
胶内酶切
四步提取法
肽质量指纹图谱
蛋白质组学
光谱分析
质量
内肽酶类
in-gel digestion
four-step extraction
peptide mass fingerprinting
proteomics
spectum analysis,mass
endopeptidases
