A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1–5 in human plasma

Age-related macular degeneration (AMD) is a leading cause of visual loss among older adults. Two variants in the complement factor H (CFH) gene, Y402H and I62V, are strongly associated with risk of AMD. CFH is encoded in Regulator of Complement Activation gene cluster in chromosome 1q32, which includes complement factor-related (CFHR) proteins, CFHR1 to CFHR5, with high amino acid sequence homology to CFH. Our goal was to build a selected reaction monitoring (SRM) assay to measure plasma concentrations of CFH variants Y402, H402, I62, and V62, and CFHR1-5. The final assay consisted of 36 peptides and 108 interference-free SRM transition ion pairs. Most peptides showed good linearity over 0.3-200 fmol/μL concentration range. Plasma concentrations of CFH variants and CFHR1-5 were measured using the SRM assay in 344 adults. Plasma CFH concentrations (mean, SE in μg/mL) by inferred genotype were: YY402, II62 (170.1, 31.4), YY402, VV62 (188.8, 38.5), HH402, VV62 (144.0, 37.0), HY402, VV62 (164.2, 42.3), YY402, IV62 (194.8, 36.8), HY402, IV62 (181.3, 44.7). Mean (SE) plasma concentrations of CFHR1-5 were 1.63 (0.04), 3.64 (1.20), 0.020 (0.001), 2.42 (0.18), and 5.49 (1.55) μg/mL, respectively. This SRM assay should facilitate the study of the role of systemic complement and risk of AMD. This article is protected by copyright. All rights reserved