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Differential protein expression in human corneal endothelial cells cultured from young and older donors.

Zhu C, Rawe I, Joyce NC - Mol. Vis. (2008)

Bottom Line: Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold.Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels.These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT

Purpose: To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors.

Methods: Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Results: Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3-10 or pH 4-7 IPG strips. Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation.

Conclusions: This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC, including differences that may affect proliferative capacity. Results indicate that HCEC from older donors exhibit reduced expression of proteins that support important cellular functions such as metabolism, antioxidant protection, protein folding, and protein degradation. These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.

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Representative two-dimensional gel images showing separation of proteins extracted from human corneal endothelial cells cultured from young and older donors. Extracted protein was pooled from five young (<30 years old; A,C) and five older donors (>50 years old; B,D). Equal amounts of protein were separated on either pH 3–10 IPG strips (A,B) or pH 4–7 IPG strips (C,D) followed by separation on 8%–16% polyacrylamide gels. Protein spots were stained with SyproRuby. Images were obtained using a ProEXPRESS Proteomic Imaging System.
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f2: Representative two-dimensional gel images showing separation of proteins extracted from human corneal endothelial cells cultured from young and older donors. Extracted protein was pooled from five young (<30 years old; A,C) and five older donors (>50 years old; B,D). Equal amounts of protein were separated on either pH 3–10 IPG strips (A,B) or pH 4–7 IPG strips (C,D) followed by separation on 8%–16% polyacrylamide gels. Protein spots were stained with SyproRuby. Images were obtained using a ProEXPRESS Proteomic Imaging System.

Mentions: Proteins were extracted from HCEC cultured from five individual donors per age group (five corneal pairs/group) and then pooled to form one sample per age group. We chose to analyze pooled samples to eliminate donor-to-donor variations in relative protein expression so as to detect specific age-related differences more easily. Besides the pooling of five individual samples, we also strictly followed a standardized 2-D separation protocol to eliminate gel-to-gel variation. Protein concentration of the two samples was determined just before the IEF first-dimensional separation. Equal amounts of protein were loaded for both gels. First- and second-dimensional separations of the two pooled samples were performed at the same time. Two-dimensional gel separations were repeated at least three times per sample, and the patterns were compared. Under these strictly controlled conditions, reproducible 2-D protein patterns were obtained using both pH 3–10 and pH 4–7 IPG strips. Figure 2A,B show typical 2-D patterns when samples were separated using pH 3–10 IPG strips. Overall, patterns from young donors (Figure 2A) were quite similar to those from older donors (Figure 2B), although several protein spots showed differences in relative density. Figure 2C,D show typical 2-D protein patterns when samples were separated using pH 4–7 IPG strips. The majority of protein spots observed using pH 3–10 IPG strips were also visible using pH 4–7 IPG strips. However, the pH 4–7 strip further separated proteins into more distinguishable spots with less streaking, thus achieving high-resolution spot separation to ensure accurate software analysis and spot-picking for protein identification. Within the maximum loading capacity of the IPG strip, more protein spots were revealed with increased protein loading, but the protein load needed to be balanced due to the tendency to induce streaking. Optimal resolution of protein spots for software analysis was obtained using a 400 μg protein load. SyproRuby was chosen for protein staining based on its excellent sensitivity and broad linear dynamic range [34].


Differential protein expression in human corneal endothelial cells cultured from young and older donors.

Zhu C, Rawe I, Joyce NC - Mol. Vis. (2008)

Representative two-dimensional gel images showing separation of proteins extracted from human corneal endothelial cells cultured from young and older donors. Extracted protein was pooled from five young (<30 years old; A,C) and five older donors (>50 years old; B,D). Equal amounts of protein were separated on either pH 3–10 IPG strips (A,B) or pH 4–7 IPG strips (C,D) followed by separation on 8%–16% polyacrylamide gels. Protein spots were stained with SyproRuby. Images were obtained using a ProEXPRESS Proteomic Imaging System.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2565687&req=5

f2: Representative two-dimensional gel images showing separation of proteins extracted from human corneal endothelial cells cultured from young and older donors. Extracted protein was pooled from five young (<30 years old; A,C) and five older donors (>50 years old; B,D). Equal amounts of protein were separated on either pH 3–10 IPG strips (A,B) or pH 4–7 IPG strips (C,D) followed by separation on 8%–16% polyacrylamide gels. Protein spots were stained with SyproRuby. Images were obtained using a ProEXPRESS Proteomic Imaging System.
Mentions: Proteins were extracted from HCEC cultured from five individual donors per age group (five corneal pairs/group) and then pooled to form one sample per age group. We chose to analyze pooled samples to eliminate donor-to-donor variations in relative protein expression so as to detect specific age-related differences more easily. Besides the pooling of five individual samples, we also strictly followed a standardized 2-D separation protocol to eliminate gel-to-gel variation. Protein concentration of the two samples was determined just before the IEF first-dimensional separation. Equal amounts of protein were loaded for both gels. First- and second-dimensional separations of the two pooled samples were performed at the same time. Two-dimensional gel separations were repeated at least three times per sample, and the patterns were compared. Under these strictly controlled conditions, reproducible 2-D protein patterns were obtained using both pH 3–10 and pH 4–7 IPG strips. Figure 2A,B show typical 2-D patterns when samples were separated using pH 3–10 IPG strips. Overall, patterns from young donors (Figure 2A) were quite similar to those from older donors (Figure 2B), although several protein spots showed differences in relative density. Figure 2C,D show typical 2-D protein patterns when samples were separated using pH 4–7 IPG strips. The majority of protein spots observed using pH 3–10 IPG strips were also visible using pH 4–7 IPG strips. However, the pH 4–7 strip further separated proteins into more distinguishable spots with less streaking, thus achieving high-resolution spot separation to ensure accurate software analysis and spot-picking for protein identification. Within the maximum loading capacity of the IPG strip, more protein spots were revealed with increased protein loading, but the protein load needed to be balanced due to the tendency to induce streaking. Optimal resolution of protein spots for software analysis was obtained using a 400 μg protein load. SyproRuby was chosen for protein staining based on its excellent sensitivity and broad linear dynamic range [34].

Bottom Line: Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold.Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels.These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT

Purpose: To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors.

Methods: Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Results: Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3-10 or pH 4-7 IPG strips. Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation.

Conclusions: This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC, including differences that may affect proliferative capacity. Results indicate that HCEC from older donors exhibit reduced expression of proteins that support important cellular functions such as metabolism, antioxidant protection, protein folding, and protein degradation. These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.

Show MeSH
Related in: MedlinePlus