Comparison of automated in-gel digest methods for femtomole level samples
Comparison of automated in-gel digest methods for femtomole level samples
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DOI：
10.1002/elps.200305615
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发表时间：
2003-10-01
期刊：
影响因子：
2.9
通讯作者：
Lee, KH
中科院分区：
文献类型：
--
作者：
Finehout, EJ;Lee, KH
自动化的凝胶内消化方法的比较低皮摩尔到飞摩尔水平的蛋白质。用考马斯亮蓝或SYPRO Ruby染色具有4pmol至120 fmol蛋白质的凝胶斑点，并使用自动化平台消化。从基质辅助激光解吸/电离质谱（MALDI-MS）分析获得的序列覆盖率和平均峰强度进行了比较。结果表明，使用乙腈提取或消化时间大于标准4小时的方法，对于低蛋白水平样品的自动消化，肽序列覆盖率没有显著增加。还显示，来自SYPRO Ruby染色的凝胶的细菌在ZipTip清除后比来自考马斯胶体蓝染色的凝胶的细菌显示出更大的改善。如果使用具有最小凝胶塞洗涤的方法，则来自SYPRO Ruby染色凝胶的肽也显示出更高的平均肽强度。
A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used.