Feasibility of two-dimensional gel electrophoresis used for proteomic analysis of human scleral fibroblasts.
OBJECTIVE
This study used two-dimensional gel electrophoresis (2-DE) to analyze protein profiles for normal human scleral fibroblasts in order to provide a baseline for future study of proteomics of the sclera in experimental conditions. In addition, differences in the presence and amount of proteins from fibroblasts isolated from the anterior or posterior sclera were analyzed.


METHODS
The fibroblasts from anterior and posterior sclera of two healthy donors were cultured separately. Proteins were extracted from the cell lines, run on 2-DE, and stained by Commassie blue R-250. The gel images were analyzed to detect differences in expression levels (at least a fivefold difference in intensity) and location of the protein spots between the anterior and posterior sclera. These protein spots were trimmed from the gels, digested with trypsin, identified by MALDI mass spectrometry, and functionally categorized with human cDNA and protein databases from NCBI.


RESULTS
The number of spots detected was 455 and 453 protein spots from the anterior and posterior scleral fibroblasts, respectively. The patterns of gel maps were very similar between the anterior and posterior sclera in each donor and between the donors in either the anterior or posterior sclera. Nine proteins showed a stronger expression in the anterior sclera compared with the posterior sclera. These proteins together with the two proteins that appeared only in the anterior sclera were mainly associated with anabolic metabolism in cells. Eight proteins showed a stronger expression in the posterior sclera, and seven of them were mainly associated with catabolic metabolism in cells. Among all 19 protein spots identified as being differentially expressed between fibroblasts originally isolated from the anterior or posterior sclera, 14 proteins had a pI (3.86-7.95) and molecular weight (23-66 kDa) consistent with those found in human from the database of NCBI and from SwissProt Entry Name.


CONCLUSIONS
The distribution and levels of expression in proteins are very similar for both the anterior and posterior sclera in vitro, with only approximately 4% of the proteins demonstrating a differential level of expression (at least fivefold) between the two segments of the sclera.
Lu
Fan
F
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China.
Zhou
Xiangtian
X
Xie
Ruozhong
R
Wu
Yaming
Y
Hu
Yongwu
Y
Zhang
Lihua
L
Li
He
H
Mao
Xinjie
X
Hu
Dannin
D
Qu
Jia
J
D000328:Adult; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism; D005240:Feasibility Studies; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D040901:Proteomics / Q000379:methods*; D012016:Reference Values; D012590:Sclera / Q000166:cytology / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
D005136:Eye Proteins