Kelly Una, Rickman Catherine Bowes, Postel Eric A, Hauser Michael A, Hageman Gregory S, Arshavsky Vadim Y, Skiba Nikolai P
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1540-5. doi: 10.1167/iovs.08-2782. Epub 2008 Nov 21.
Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient's blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes.
A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes.
The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402.
A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.
补体因子H（CFH）基因变异在不同人群中与年龄相关性黄斑变性（AMD）紧密相关。在CFH众多非同义编码变异中，有两个与AMD风险增加关联最为紧密：异亮氨酸62突变为缬氨酸（I62V）以及酪氨酸402突变为组氨酸（Y402H）。检测患者血液中的这些变异对于AMD风险评估和疾病预后至关重要。然而，传统的基因分析方法无法用于检测某些人血浆及其他不含DNA的生物体液中的CFH同种异型。目的是开发一种基于蛋白质的CFH同种异型检测方法。
采用一步亲和富集CFH、凝胶分离以及对跨越62位和402位氨基酸的CFH肽段进行质谱鉴定相结合的方法来鉴定个体CFH同种异型。
基于使用基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-TOF）分别鉴定出质量为1148.59 Da、1162.60 Da、2031.88 Da和2057.88 Da的胰蛋白酶肽段，可靠地检测到了CFH异构体V62、I62、H402和Y402。由于62位和402位的变异，这些肽段在不同CFH样品质谱图中的存在或缺失模式与CFH的所有九种基因型密切相关。
已开发出一种快速灵敏的方法，用于通过质谱检测人血浆样品中CFH的V62...
