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-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.
Golaz
O
O
Clinical Chemistry Laboratory, Geneva University Hospital, Switzerland. golaz/dminov1.hcuge.ch
Wilkins
M R
MR
Sanchez
J C
JC
Appel
R D
RD
Hochstrasser
D F
DF
Williams
K L
KL
D000595:Amino Acid Sequence; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005069:Evaluation Studies as Topic; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis* / Q000145:classification; D015203:Reproducibility of Results; D012984:Software
D011506:Proteins