Technical aspects of gel-based proteomics designed for elucidating an aryl hydrocarbon receptor complex.
The identification of proteins by mass spectrometry has revolutionalized the basic method of identifying proteins constituting an intracellular unit or network for certain biological functions. The gel-based strategy following immunoprecipitation was applied to elucidating proteins associated with the aryl hydrocarbon receptor (AhR). Two hundred femtomoles of AhR was recovered from approximately 2 x 10(7) HepG2 cells by immunoprecipitation and was sufficient for identification by peptide mass fingerprinting. Possible candidates for the AhR-associated proteins were also identified. Improvements of the current strategy to increase the overall sensitivity tenfold are required to clarify the AhR complex in full detail. For example, a combination of trypsin and Achromobacter protease I for in-gel digestion allows the number of missed cleavage sites to be set at zero for database searching, thereby reducing random matches and facilitating identification. There is also room for improvement in each step of sample preparation prior to mass spectrometry.
Wada
Yoshinao
Y
Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan. waday@mch.pref.osaka.jp
Nakano
Norihiko
N
D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D005782:Gels; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis*; D012697:Serine Endopeptidases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D005782:Gels; D018336:Receptors, Aryl Hydrocarbon; D012697:Serine Endopeptidases; D014357:Trypsin; C036813:lysyl endopeptidase