Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics.
Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.
Hathout
Yetrib
Y
Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA. yhathout@cnmcresearch.org
Flippin
Jessica
J
Fan
Chenguang
C
Liu
Pinghu
P
Csaky
Karl
K
D000368:Aged; D000369:Aged, 80 and over; D002455:Cell Division; D002478:Cells, Cultured; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization