Metabolic Labeling of Human Primary Retinal Pigment Epithelial Cells for Accurate Comparative Proteomics

Abstract

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.

Quantitative Proteomics Reveals an Altered Cystic FibrosisIn VitroBronchial Epithelial Secretome

“…We hypothesized that mutant CFTR results in altered protein levels in the CF airway epithelial secretome under constitutive conditions at ALI in the absence of infection and inflammatory cells. To test this, we used stable isotope labeling with amino acids in cell culture (SILAC), a highly accurate and quantitative mass spectrometry (MS)-based proteomics technique (28), and three CF (DF508/ DF508) and three non-CF life-extended HBE cell lines that could be passaged several times (29) to ensure .98% amino acid incorporation for quantitative secretome analyses.…”

“…Before seeding at ALI, passage 12 UNCCF cells were labeled by SILAC for two passages (28). Cells were proliferated in bronchial epithelial growth medium (Lonza) containing "heavy" ("labeled") 13 C 6 -Arg (2 mM) and 13 C 6 , 15 N 2 -Lys (0.2 mM) (Cambridge Isotopes, Andover, MA).…”

Directional Secretomes Reflect Polarity-Specific Functions in an In Vitro Model of Human Bronchial Epithelium

“…Proteins were fractionated by gel electrophoresis; bands were excised and trypsin-digested. The resulting peptides were analyzed by LC-MS/ MS as previously described (24)(25)(26). Further details are provided in the online supplement.…”

Application of proteomic technology in eye research: a mini review

“…Proteomic study has also been carried out to localise the cellular retinaldehyde‐binding protein in the apical membrane of the RPE for retinoid processing and trafficking 110 . Recently, by combining metabolic labelling and proteomic approach, differentially expressed proteins, which are involved in cell proliferation, protein synthesis, actin‐remodelling and differentiation in human RPE, were studied 111 . The studies provided insights into the normal physiology of this important cellular layer.…”