Factor H and related proteins in AMD

Age-related macular degeneration (AMD) is a common form of adult blindness. Risk of developing AMD is strongly linked to genetics, in particular at the Regulators of Complement Activation (RCA) locus on Chr1, which contains 6 genes, Complement Factor H (CFH) and Complement Factor H-related 1-5 (CFHR1-5). These encode seven distinct proteins, since CFH gives rise to both a full length Factor H (FH) and a small splice variant, FHL-1 protein. These proteins act as fluid-phase co-factors for the breakdown of C3b and hence regulation of the complement cascade. Many of these SNPs are intronic; there is speculation that they modify the systemic levels of these proteins, causing an imbalance on complement regulation which impacts AMD development. Since FH, Factor H-related (FHR1-5) and FHL-1 proteins exhibit a high degree of homology, there is no antibody-based assay available for the simultaneous quantification of all seven related proteins. Here we describe a novel mass spectrometry-based assay for the simultaneous detection and quantification of all seven highly homologous human complement proteins; FH, FHR1, FHR2, FHR3, FHR4, FHR5 and FHL-1, in plasma. Since proteolysis using trypsin is not amenable to the generation of reliable proteotypic peptides for all seven proteins, we utilised Endoproteinase Glu-C (V8 Protease) as an alternative. We were able to generate unique signature peptides for all seven of the target proteins in a simple protocol which requires no pre-fractionation or clean-up stages during sample preparation. The method described is shown to be capable of the robust, simultaneous detection and relative quantification of all seven proteins with linearity and limits of detection appropriate to physiological levels. Once validated, the method was successfully applied to a clinical set of AMD control samples and compared to an existing FH antibody-based approach.

年龄相关性黄斑变性（Age-related macular degeneration, AMD）是成人失明的常见病因。AMD的发病风险与遗传因素显著相关，尤其是位于1号染色体的补体激活调节因子（Regulators of Complement Activation, RCA）位点，该位点包含6个基因：补体因子H（Complement Factor H, CFH）以及补体因子H相关蛋白1-5（CFHR1-5）。由于CFH可同时编码全长因子H（FH）与一种小型剪接变体FHL-1蛋白，因此上述基因共编码7种不同的蛋白质。这些蛋白作为C3b降解的液相辅因子，进而调控补体级联反应。其中多数单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）位于内含子区域；有研究推测，这些SNPs可改变这些蛋白的全身表达水平，引发补体调控失衡，进而影响AMD的发生发展。由于FH、FHR1-5与FHL-1蛋白具有高度同源性，目前尚无能够同时定量这7种相关蛋白的基于抗体的检测手段。本文报道了一种基于质谱的新型检测方法，可在血浆样本中同时检测并定量这7种高度同源的人补体蛋白：FH、FHR1、FHR2、FHR3、FHR4、FHR5及FHL-1。由于使用胰蛋白酶水解无法为全部7种蛋白生成可靠的蛋白特征肽段，我们采用了Glu-C内切蛋白酶（Endoproteinase Glu-C, V8 Protease）作为替代方案。我们通过一套简单的样品制备流程即可为全部7种靶蛋白生成独特的特征肽段，该流程无需预分级或纯化步骤。实验结果表明，本方法可实现7种蛋白的稳定、同时检测与相对定量，其线性范围与检测限均适配生理水平的检测需求。经验证后，该方法成功应用于一组AMD对照临床样本，并与现有的基于抗体的FH检测方法进行了对比。