Protein expression in a transformed trabecular meshwork cellline: proteome analysis

[摘要] Purpose: Characterization of the human trabecular meshwork (TM)proteome is hindered by the small mass of intact tissue and the slowgrowth of cultured cell strains. We have previously characterized atransformed TM cell strain (GTM3) that demonstrates many of the sameprotein expression and cell signaling systems of nontransformed cellstrains. The aim of this study was to initiate a proteomic survey ofGTM3 cells as the initial step toward characterization of the completehuman TM proteome.Methods: GTM3 cells were cultured to confluence, harvested andsolubilized in urea/Nonidet. The protein extract (600 μg) was focusedin immobilized isoelectric focusing (IEF) strips, separated by 10% SDSPAGE, and visualized with colloidal Coomassie Blue. Spots of interestwere excised, destained, and the contained proteins subjected to in-gelreduction, derivatization, and tryptic digestion. Tryptic peptides wereextracted and analyzed by electrospray LC/MS/MS. Protein identificationwas made using the TurboSequest search algorithm and a recent version ofthe nonredundant human protein database downloaded from the NationalCenter for Biotechnology Information (NCBI).Results: Eighty-seven (87) primary proteins and 93 variants of theseproteins were identified. A website was created (TMproteome) that combines data such as graphic spot location within thegel, peptide sequence, apparent and calculated pI, apparent andcalculated mass, percentage of coverage, and protein informatic websitelinks.Conclusions: Proteomic analysis of a transformed human TM cell linehas been initiated combining preparative two-dimensional PAGEseparation, LC/MS/MS analysis of major proteins, and bioinformaticcataloging of the data. Further investigation of data from thetransformed cell strain will be used in a comparative fashion for spotidentification of analytical proteomic gels of human TM tissue andcultured normal cells. These initial data will form the base from whichthe characterization of protein expression in the normal andglaucomatous TM can be accomplished.

[学科分类] 生物化学/生物物理