Golaz O, Wilkins M R, Sanchez J C, Appel R D, Hochstrasser D F, Williams K L
Clinical Chemistry Laboratory, Geneva University Hospital, Switzerland. golaz/dminov1.hcuge.ch
Electrophoresis. 1996 Mar;17(3):573-9. doi: 10.1002/elps.1150170328.
Expression of different genomes can be studied by high-resolution two-dimensional electrophoresis (2-D PAGE). To help these studies, two-dimensional reference maps of different biological tissues and fluids have been built and can be found in the SWISS-2DPAGE database, accessible via the World Wide Web network on the ExPASy molecular biology server. Different techniques were used to identify the polypeptides. At the present time, the method considered to be the fastest and the most cost-effective is amino acid composition analysis (AAC). Proteins, transferred onto polyvinylidene (PVDF) membranes, were submitted to vapor-phase hydrolysis, derivatized with 9-fluorenylmethyl chloroformate (FMOC) and separated on an ODS-Hypersil column. Identification was obtained by using the program 'AACompIdent' available from ExPASy. In this work, different experimental parameters, such as contamination, reproducibility and accuracy, have been assessed. First, it has been found that a major source of contamination was human keratin. Next, amino acids have been classified into 'reliable' and 'nonreliable'. Accordingly, 'bias' and 'weights' were defined for each amino acid, which could be set in the 'AACompIdent' program. Finally, examples of identification, including the use of Edman degradation sequence tagging, are described.
不同基因组的表达可通过高分辨率二维电泳（2-D PAGE）进行研究。为辅助这些研究，已构建了不同生物组织和体液的二维参考图谱，可在SWISS-2DPAGE数据库中找到，该数据库可通过ExPASy分子生物学服务器上的万维网网络访问。使用了不同技术来鉴定多肽。目前，被认为是最快且最具成本效益的方法是氨基酸组成分析（AAC）。转移到聚偏二氟乙烯（PVDF）膜上的蛋白质进行气相水解，用9-芴甲基氯甲酸酯（FMOC）衍生化，并在ODS-Hypersil柱上分离。通过使用ExPASy提供的“AACompIdent”程序进行鉴定。在这项工作中，评估了不同的实验参数，如污染、重现性和准确性。首先，发现主要污染源是人类角蛋白。其次，氨基酸被分为“可靠”和“不可靠”两类。相应地，为每个氨基酸定义了“偏差”和“权重”，可在“AACompIdent”程序中设置。最后，描述了鉴定实例，包括使用埃德曼降解序列标签。
