Methods Of Ionization In Mass Spectroscopy

蛋白质结构分析方法正在蛋白质组学独立和合作研究项目中得到改进、应用和测试。一个自动化的二维液相色谱系统已被进一步改进，用于与亚微升微柱兼容的低流量，用于使用纳米电喷雾电离质谱法分析复杂的肽混合物。该系统使用强阳离子交换色谱法进行分馏，然后在单个反相柱上进行梯度洗脱。该系统已被测试与可溶性蛋白质从酵母和能够识别数百种蛋白质在所得的复杂提取物。已经进行了微喷雾2D-LC系统的广泛评价，以确定最佳的系统容量和分馏条件。LC-MS/MS肽分析常规地产生大数据集，从而使用自动化解释程序产生鉴定的肽和蛋白质的长列表。得到的文件包含相关的实验信息，但是当分析多个文件时，特别是当需要总结报告时，这些结果的后续排序、整理和比较构成了重要的任务。我们合作开发了软件，便于多个文件的解释和比较。Perl程序（DBParser）从专有搜索程序平面文件中检索输出，并将数据存储在新的数据库中。该程序生成用户友好的html输出报告的排序和比较的肽/蛋白质列表，可用于后续分析。基于CGI的图形用户界面允许使用Web浏览器通过网络执行DBParser，以方便多个实验室使用。

Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. An automated two dimensional liquid chromatographic system has been further improved for lower flow compatible with sub-microliter microcolumns for the analyses of complex mixtures of peptides using nano-electrospray ionization mass spectrometry. The system fractionates using strong cation exchange chromatography, followed by gradient elution on a single reverse phase column. The system has been tested with soluble proteins from yeast and is capable of identifying hundreds of proteins in the resulting complex extract. Extensive evaluation of the microspray 2D-LC system has been performed to define optimum system capacity and fractionation conditions. LC-MS/MS peptide analyses routinely generate large data sets leading to long lists of identified peptides and proteins using automated interpretation programs. The resulting files contain relevant experimental information, but subsequent sorting, collation, and comparison of these results pose significant tasks when analyzing multiple files, especially when summary reports are desired. We have developed software collaboratively that facilitates multiple file interpretation and comparison. A perl program (DBParser) retrieves output from proprietary searching program flat files and stores data in a new database. The program generates user-friendly html output reports of the sorted and compared peptide/protein lists which can be used for subsequent analysis. A CGI-based graphical user interface allows execution of DBParser over a network using a web browser to facilitate its use by multiple laboratories.