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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >THE ANALYSIS OF MYOCARDIAL PROTEINS BY INFRARED AND ULTRAVIOLET LASER DESORPTION MASS SPECTROMETRY
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THE ANALYSIS OF MYOCARDIAL PROTEINS BY INFRARED AND ULTRAVIOLET LASER DESORPTION MASS SPECTROMETRY

机译：红外和紫外激光解吸质谱法分析心肌蛋白

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摘要

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer, In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required. [References: 20]
机译：讨论了使用红外（IR）和紫外（UV）基质辅助激光解吸（MALDI）质谱分析二维（2-D）聚丙烯酰胺凝胶电泳（PAGE）分离的心肌蛋白。将蛋白质电印迹到FluoroTrans聚偏二氟乙烯（PVDF）膜上，以便于通过MALDI进行分析，这是同时提取大量蛋白质的最有效方法。一旦在FluoroTrans膜上，IR-MALDI即可从选定的蛋白质斑点中获得光谱，但UV MALDI无法获得有用的信号。从分析的50个斑点中的46个产生光谱，分析的蛋白质质量为13至82 kDa，等电点（pI）为4.7-7.8。对于那些先前已表征的蛋白质斑点，其序列和翻译后修饰数据均已知，IR-MALDI数据在预期质量的正负0.5％之内。一些斑点包含一个以上的蛋白质信号，这说明可以从MALDI获得更多的信息，但也暗示了用于分离大量蛋白质的2-D凝胶分辨率的限制。尝试使用特定的蛋白酶消化蛋白质并通过MALDI分析在膜上产生肽质量指纹图谱，无论是使用IR还是UV激光都没有成功。从膜中提取肽，并通过UV MALDI轻松分析其肽谱。用IR-MALDI进行肽消化的数据很差，可能是由于消化缓冲液对基质的抑制作用。为了从印迹的蛋白质中获得最大的信息量，IR和UV MALDI都是必需的。 [参考：20]

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