Preparation and analysis of proteins and peptides using MALDI TOF/TOF mass spectrometry.
Preparation and analysis of proteins and peptides using MALDI TOF/TOF mass spectrometry.
复制标题
DOI：
10.1002/0471140864.ps1613s63
复制
发表时间：
2011-02-01
影响因子：
--
通讯作者：
Gorman, Jeffrey J
中科院分区：
文献类型：
--
作者：
Dave, Keyur A;Headlam, Madeleine J;Gorman, Jeffrey J
基质辅助激光解吸/电离飞行时间/飞行时间质谱仪(MALDI-TOF/TOF-MS)是多肽和蛋白质分析的重要工具。特别有用的特性包括高灵敏度、快速数据采集、易用性和强大的仪器设备。虽然MALDI对缓冲液和其他杂质的容忍度相对较高，但通过去除样品中的非分析物成分可以显著提高灵敏度。因此，对样品进行处理，去除缓冲液和杂质，可以大大提高MALDI实验结果的质量。本单元描述了用于酶消化、MALDI靶板制备、薄层基质制备、目标样品清理以及分析物和基质的毛细管高效液相色谱-MALDI共斑点的优化程序。还描述了在胰酶消化前用MALDI-TOF/TOF-MS分析膜上蛋白的程序。其中一些程序也适用于来自二维(2-D)凝胶的蛋白质斑点。还为MS和MS/MS光谱的获取和解释提供了指导。
Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation. Although MALDI is relatively tolerant to buffers and other impurities, substantial sensitivity enhancement can be achieved through removal of non-analyte components of samples. Therefore, sample processing to remove buffers and impurities can greatly improve the quality of results obtained by MALDI experiments. This unit describes optimized procedures for enzymatic digestion, preparation of MALDI target plates, thin layer matrix preparation, on-target sample cleanup, and capillary HPLC-MALDI co-spotting of analyte and matrix. Procedures are also described for analysis of on-membrane proteins by MALDI-TOF/TOF-MS before tryptic digestion. Some of these procedures are also applicable to protein spots from two-dimensional (2-D) gels. Guidance is also provided for acquisition and interpretation of MS and MS/MS spectra.