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A more recent version of this article appeared on April 1, 2009
 (Investigative Ophthalmology and Visual Science. )
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2782
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Article

Rapid and sensitive method for detection of Y402, H402, I62 and V62 variants of Complement Factor H in human plasma samples using mass spectrometry

Una Kelly 1, Catherine Bowes Rickman 1, Eric Postel 1, Michael A Hauser 1, Gregory S. Hageman 2, Vadim Arshavsky 1, and Nikolai P Skiba 1*

1 Ophthalmology, Duke University, Durham, North Carolina, United States
2 Ophthalmology & Visual Sciences, University of Iowa - Center for Macular Degeneration, Iowa City, Iowa, United States

* To whom correspondence should be addressed. E-mail: nikolai.skiba{at}duke.edu.


  Abstract

Purpose. Variations in the complement factor H (CFH) gene are tightly associated with age-related macula degeneration (AMD) across diverse populations. Of the many non-synonymous coding variants in CFH, two are most strongly associated with increased AMD risk: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in patients 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. Our purpose was to develop a protein-based method of detecting CFH allotypes. Methods. To identify individual CFH allotypes, we used 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. Results. We reliably detected CFH isoforms V62, I62, H402 and Y402 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 9 genotypes of CFH as a result of variations at positions 62 and 402. Conclusions. We developed a rapid and sensitive method 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.

Key Words: age-related macular degeneration, complement factor H, mass spectrometry






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