Finehout Erin J, Lee Kelvin H
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Electrophoresis. 2003 Oct;24(19-20):3508-16. doi: 10.1002/elps.200305615.
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.
本文介绍了用于低皮摩尔至飞摩尔水平蛋白质的自动化凝胶内消化方法的比较。含有4皮摩尔至120飞摩尔蛋白质的凝胶斑点用考马斯胶体蓝或SYPRO Ruby染色，并使用自动化平台进行消化。比较了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。结果表明，使用乙腈萃取或消化时间大于标准4小时的方法，对于低蛋白质水平样品的自动化消化，肽序列覆盖率没有显著增加。还表明，SYPRO Ruby染色凝胶的消化物在ZipTip净化后比考马斯胶体蓝染色凝胶的消化物有更大的改善。如果使用凝胶块洗涤最少的方法，SYPRO Ruby染色凝胶的消化物也显示出更高的平均肽强度。
