IDENTIFICATION OF PROTEINS BY THEIR AMINO-ACID-COMPOSITION - AN EVALUATION OF THE METHOD

Expression of different genomes can be studied by high-resolution two- dimensional electrophoresis (2-D PAGE). To help these studies, two-dim ensional reference maps of different biological tissues and fluids hav e 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 cos t-effective is amino acid composition analysis (AAC). Proteins, transf erred onto polyvinylidene (PVDF) membranes, were submitted to vapor-ph ase 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 wor k, different experimental parameters, such as contamination, reproduci bility 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 s et in the 'AACompIdent' program. Finally, examples of identification, including the use of Edman degradation sequence tagging, are described .