TY - JOUR
T1 - Quantitative analysis of hydroxyapatite-binding plasma proteins in genotyped individuals with late-stage age-related macular degeneration
AU - Arya, Swati
AU - Emri, Eszter
AU - Synowsky, Silvia A.
AU - Shirran, Sally L.
AU - Barzegar-Befroei, Neda
AU - Peto, Tunde
AU - Botting, Catherine H.
AU - Lengyel, Imre
AU - Stewart, Alan J.
N1 - We are also grateful to Fight for Sight for financial support (project grant to A.J.S and I.L.; grant ref.: 1586/1587). This research was also supported by “Eye-Risk” a European Union’s Horizon 2020 research and innovation program (grant ref.: 634479), Bill Brown Charitable Trust, MEH Special Trustees and Mercer Fund (I.L). This work was also supported by the Wellcome Trust (grant ref.: 094476/Z/10/Z) for funding the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews.
PY - 2018/7
Y1 - 2018/7
N2 - Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (p<0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation.
AB - Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (p<0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation.
KW - Drusen
KW - Sub-retinal pigment epithelial deposits
KW - Mineral-protein interactions
KW - Retinal disease
KW - Quantitative proteomics
UR - https://www.sciencedirect.com/science/article/pii/S0014483517307819#appd001
U2 - 10.1016/j.exer.2018.03.023
DO - 10.1016/j.exer.2018.03.023
M3 - Article
SN - 0014-4835
VL - 172
SP - 21
EP - 29
JO - Experimental Eye Research
JF - Experimental Eye Research
ER -