A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

中文翻译：

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2D凝胶蛋白的鉴定：MALDI / TOF肽质谱图与mu LC-ESI串联质谱的比较。

使用MALDI-TOF肽质量图谱（PMM）和mu LC-MS / MS，通过二维凝胶电泳从两个完全测序的古生甲烷菌（Methanococcus jannaschii）和激烈热球菌（Pyrococcus furiosus）分离的总共162个蛋白斑点的蛋白质鉴定比较分析是提出了。通过mu LC-MS / MS，分析出的100％凝胶斑点成功地与两个相应的开放阅读框数据库中的预测蛋白质相匹配，而MALDI-TOF PMM鉴定出其中的97％。PMM的高成功率来自样品的ZipTip（C18）脱盐/浓缩以及多个PMM搜索参数的优化，包括25 ppm平均质量容差和两个不同蛋白质分子量搜索窗口的应用。通过使用这种策略，低分子量（< 可以用少于5个肽段匹配明确鉴定23 kDa）蛋白。9％的斑点被鉴定为包含多种蛋白质。通过使用mu LC-MS / MS，分析的斑点中有50％被鉴定为包含多种蛋白质。在已鉴定的整个蛋白质质量范围内，μLC-MS / MS表现出比MALDI-TOF PMM更好的蛋白质序列覆盖率。MALDI-TOF和PMM产生了独特的肽分子量匹配，而μLC-MS / MS却无法鉴定出该分子量。通过将氨基酸序列修饰结合到数据库搜索中，从这两种互补电离方法获得的组合序列覆盖率超过了162％的70％的50％。