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,1,2 Eric A. Postel,1 Michael A. Hauser,1,3,4 Gregory S. Hageman,5 Vadim Y. Arshavsky,1,6 and Nikolai P. Skiba1

PURPOSE. Variations in the complement factor H (CFH) gene are omplement factor H (CFH) is an abundant plasma glycopro- tightly associated with age-related macular degeneration Ctein that is the main soluble inhibitor of the alternative com- (AMD) across diverse populations. Of the many nonsynony- plement pathway. Variations in the CFH gene have been impli- mous coding variants in CFH, two are most strongly associated cated as one of the strongest genetic risk factors for early-stage with increased risk of AMD: isoleucine 62 to valine (I62V) and age-related macular degeneration (AMD), accounting for up to tyrosine 402 to histidine (Y402H). Detection of these variations 50% of the population-attributable risk percentage.1–4 Of the in a patient’s blood is important for a risk assessment of AMD many nonsynonymous coding variants in CFH, two are strongly and disease prognosis. However, traditional methods of ge- associated with increased AMD risk: isoleucine 62 to valine (I62V) netic analysis cannot be used for measuring CFH allotypes in and tyrosine 402 to histidine (Y402H).4 It has been also reported some sources of human plasma and other biological fluids not that the same variations in the CFH gene are associated with containing DNA. The purpose was to develop a protein-based dense deposit disease (DDD; known formerly as membranopro- method of detecting CFH allotypes. liferative glomerulonephritis type II), suggesting that dysregula- tion of the complement alternative pathway is important in this METHODS. A combination of a single-step affinity enrichment of 4,5 CFH, gel separation, and mass spectrometry identification of disease pathophysiology, as well. Identification of persons car- the CFH peptides spanning amino acids at positions 62 and 402 rying CFH variants associated with these diseases would allow was used to identify individual CFH allotypes. clinicians to provide an accurate risk assessment and develop strategies to reduce effects of other extrinsic factors. The ability to RESULTS. The CFH isoforms V62, I62, H402, and Y402 were identify CFH variants in human blood or plasma also facilitates reliably detected based on identification of tryptic peptides purification of CFH variants for biochemical studies aimed at with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and understanding how these amino acid substitutions effect a change 2057.88 Da, respectively, using MALDI-TOF-TOF. The pres- in biochemical properties of the CFH that contribute to risk for ence or absence pattern of these peptides in mass spectra of AMD and DDD. different CFH samples robustly correlated with all nine geno- Current methods of detecting CFH allelic variants rely pri- types of CFH, as a result of variations at positions 62 and 402. marily on PCR-based DNA genotyping of patient’s blood, CONCLUSIONS. A rapid and sensitive method has been developed whereas testing of CFH protein variants in human plasma is still for detection of V62, I62, H402, and Y402 variants of CFH in in its earliest stage of development. Recently, a method was human plasma samples using mass spectrometry. This method described for measurement of CFH Y402H variants in plasma can be used in clinical laboratories equipped with a basic using variant-specific monoclonal antibodies.6 This method is inexpensive mass spectrometer capable of performing peptide limited by availability of the monoclonal antibody and allows fingerprinting. (Invest Ophthalmol Vis Sci. 2009;50:1540–1545) testing for only a single CFH variant, Y402H. Herein, we de- DOI:10.1167/iovs.08-2782 scribe a mass spectrometry (MS)-based method for rapid and sensitive detection of Y402, H402, I62, and V62 variants of CFH in human plasma samples. For detection by this method, plasma CFH is first enriched on a heparin-agarose column, From the Departments of 1Ophthalmology, 2Cell Biology, 3Medi- 6 4 separated on a polyacrylamide gel, in-gel cleaved with trypsin, cine, and Pharmacology and Cancer Biology, and the Center for and finally analyzed by MS. The reproducibility and specificity Human Genetics, Duke University Medical Center, Durham, North Carolina; and the 5Department of Ophthalmology and Visual Sciences, of this method was validated with DNA-genotyped plasma University of Iowa, Iowa City, Iowa. samples and demonstrated 100% accuracy in identifying all Supported by The Foundation Fighting Blindness (CBR); in part by nine genotypes of CFH resulting from combinations of the core grants from the National Eye Institute (NEI; P30 EY005722) and polymorphisms at amino acid positions 62 and 402. Research to Prevent Blindness to the Duke Eye Center; National Insti- tutes of Health Grant R24 EY017404 (GSH); and funds from NEI Grant EY012118 provided by Margaret Pericak-Vance (University of Miami) METHODS and Jonathan Haines (Vanderbilt University), which funded a portion of the samples used in this work. Plasma Samples Submitted for publication August 28, 2008; revised November 4, The plasma samples used in this study were obtained from the cohort 2008; accepted Januray 27, 2009. of genotyped blood samples at Duke University2 and the University of Disclosure: U. Kelly, None; C. Bowes Rickman, None; E.A. 4 Postel, None; M.A. Hauser, None; G.S. Hageman, Optherion, Inc. Iowa to establish the initial association of factor H and the risk of (I); V.Y. Arshavsky, None; N.P. Skiba, None developing AMD. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Plasma Factor H Enrichment ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. and PAGE Separation Corresponding author: Nikolai Skiba, Department of Ophthalmol- ogy, Duke University Medical Center, Albert Eye Research Institute Immobilized heparin-agarose (50 ␮L; Pierce, Rockford, IL) was added to a Room 5006, Box 3802, Erwin Road, Durham, NC 27710; column (Handi-spin; Pierce) and the beads washed twice with 300 ␮Lof [email protected]. 10 mM sodium phosphate buffer pH 7.4. Plasma (30 ␮L) was diluted with

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90 ␮L of the same buffer before it was added to the beads. The columns were capped and rotated at room temperature for 15 minutes and cen- trifuged, and the beads washed three times. CFH was eluted with 40 ␮L buffer containing 500 mM NaCl. In some cases, it was further enriched by gel-filtration on an FPLC column (16/10 Superose; GE Healthcare, Piscat- away, NJ). Typically, 1 ␮L of crude plasma, 20 ␮L of eluate from a heparin-Sepharose column, and 0.5 to 1 ␮g of CFH collected in gel filtration eluate were applied to a 10% Bis-Tris Precast gel (Criterion XT; FIGURE 1. Coomassie stained SDS- Bio-Rad, Hercules, CA) for SDS-PAGE. Known amounts of commercially PAGE gel of various CFH prepara- available CFH protein standard (Complement Technology, Tyler, TX) tions. Lane 1: commercial CFH stan- were run on the same gel. After electrophoresis, the gel was washed in dard (0.5 ␮g); lane 2:1␮L of plasma water and stained with Coomassie blue reagent (Gel Code Blue Stain; obtained from an I62/Y402 individual; lane 3:20␮L of CFH-containing Pierce) for 1 hour, followed by a 30-minute rinse in water. Bands, running eluate from heparin-agarose column at the same position as the CFH standard, were cut from the gel, reduced (single step enrichment); and lane 4: with 10 mM DTT, modified at cysteine residues with iodoacetoamide and CFH purified on a heparin-agarose trypsinized according to the manufacturer’s protocol (In-Gel Tryptic Di- column followed by a gel filtration gestion Kit; Pierce). column (0.8 ␮g).

FIGURE 2. MS/MS spectra of precursors 1148.59 (A) and 1162.60 (B) conﬁrming the sequence identity of CFH peptides SLGNVIMVCR (peptide 1a) and SLGNIIMVCR (peptide 1b), respectively.

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MS Analysis enrichment procedures, thanks to the ability of this method to identify hundreds of proteins in crude mixtures. To establish The extracted peptides were dissolved in 5 ␮L of 50% acetonitrile and the applicability of MS to identification of the CFH isoforms in 0.1% trifluoroacetic acid containing 5 mg/mL ␣-4-hydroxycinnamic acid (Sigma-Aldrich) as a matrix for MALDI MS. The peptide–matrix human blood, we obtained plasma samples containing four mixture (0.5 ␮L) was loaded onto a 192-spot MALDI plate and analyzed different combinations of the amino acids at positions 62 on a MALDI-TOF-TOF system (4700 Proteomic Analyzer; Applied Bio- (I62V) and 402 (Y402H) in CFH. The four combinations we systems, Foster City, CA). A combination of peptide mass fingerprint- used (IIYY, VVHH, VVYY, and VIYH) were each enriched, ing and precursor fragmentation analysis was performed by using the either on heparin-agarose or on heparin-agarose followed by Mascot algorithm to search the NCBInr human database (http:// gel filtration chromatography. Typically, these procedures www.ncbi.nlm.nih.gov/sites/entrez?dbϭprotein; provided in the pub- yielded between 0.1 and 1 ␮g of CFH from 30 ␮L of plasma, lic domain by National Center for Biotechnology Information, Be- which ran as a distinct band of ϳ190 kDa (Fig. 1). thesda, MD). Search parameters were the following: precursor toler- The identity of the 190-kDa band as CFH was established ance, 50 ppm; MS/MS fragment tolerance, 0.5 Da; fixed modification, using the MALDI-TOF-TOF analysis, which gave an identifica- carbimidomethyl; variable modification, methionine oxidation; and tion confidence interval of 100% for control commercial CFH number of missing cleavages, 1. standards and the CFH samples enriched on heparin-agarose columns or both heparin-agarose and gel filtration columns. However, when analysis of plasma samples was performed RESULTS without any CFH enrichment, confident identification of CFH MS provides a rapid and reliable method of identifying proteins protein could not be consistently achieved. Typical sequence and their isoforms. It does not require extensive purification of coverage for CFH identification ranged from 30% to 50% for proteins of interest from biological samples, but rather simple individual samples, with the number of mass values matched to

FIGURE 3. MS/MS spectra identifying the precursors 2031.88 (A) and 2057.88 (B) as CFH peptides CYFPY- LENGYNQNHGR (peptide 2a) and CYFPYLENGYNQNYGR (peptide 2b), respectively.

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predicted peptides ranging from 33 to 49 of a total of 79 To determine whether we could distinguish the Y402, theoretical peptides. The exclusion of predicted CFH tryptic H402, V62, and I62 variants of CFH in human plasma samples, peptides with masses beyond the MS identification range of we analyzed gel bands corresponding to CFH from genotyped 800 to 4000 Da decreased the number of predicted tryptic human plasma samples (Fig. 1). Figure 4 shows mass peaks peptides to 70. In addition, the CFH glycoprotein contains 1148.59 Da (referred to from now on as peak 1148) and eight N-glycosylation sites,7 four of which are located within 1162.60 Da (peak 1162) corresponding to peptides 1a and 2b, the tryptic peptides in the mass range of 800 to 4000 Da. respectively, which were observed in the samples with the Consistently, none of these peptides was identified by MS. genotypes VV62 (Fig. 4A), II62 (Fig. 4B) and VI62 (Fig. 4C). Therefore, the total number of theoretically identifiable pep- The appearance of 1148 peak corresponded to the VV or VI tides was 66 and the identification rate was up to 74%. The genotype, whereas the appearance of peak 1162 correlated absence of mass peaks corresponding to the remaining 17 with the genotype corresponding to CFH 62VI or 62II. The predicted CFH tryptic peptides can be explained by not yet peak intensity and signal-to-noise ratio for both peaks were identified posttranslational modifications, weak ionization, or sufficiently high to allow a reliable detection of these peaks in cleavage constraints for trypsin. Of interest, protein scores for all the samples tested. Peak 1148 was absent in samples with a CFH enriched by either one or two column steps were similar II62 genotype (Fig. 4B), and peak 1162 was absent in samples (data not shown), despite a noticeable enrichment of the with VV62 genotype (Fig. 4A). Figure 5 shows mass peaks of protein after two column steps compared with the one column 2031.88 (peak 2031) and 2057.88 (peak 2057) Da, correspond- procedure (Fig. 1). The intensities of mass peaks were also ing to peptides 2a and 2b, respectively. The peptide 2a, peak comparable for CFH samples after a one- or two-step purifica- 2031 was detected in samples with genotypes corresponding tion (data not shown). Therefore, the single step purification to the CFH HH402 and HY402 variants (Figs. 5A, 5C), while the strategy on heparin-agarose was sufficient for reliable CFH peptide 2b, peak 2057 was detected in samples that were detection by MS analysis. YY402 and HY402 (Figs. 5B, 5C). The peptide 2a was absent in Fortunately, both peptides containing the amino acid residues varying among the CFH isoforms displayed intense mass samples with the YY variant and the peptide 2b was absent in peaks, whose identities were further confirmed by the MS/MS samples with the HH variant. analysis. Figure 2 illustrates the identification of the V62 and To establish the detection reliability of the CFH isoforms I62 CFH variants based on the MS/MS spectra of the peptides (V62, I62, H402, and Y402) based on identification of the SLGNV62IMVCR (1148.59 Da; Fig. 2A, peptide 1a) and peptides 1a, 1b, 2a, and 2b, we analyzed human plasma sam- SLGNI62IMVCR (1162.60 Da; Fig. 2B, peptide 1b). Simi- ples of all nine CFH genotypes (VVHH, VIHH, IIHH, VVHY, larly, the identities of the H402 and Y402 CFH variants VIHY, IIHY, VVYY, VIYY, and IIYY). In each case, the appear- were established based on the mass peaks of peptides ance of the 1148, 1162, 2031, and 2057 mass peaks reflected CYFPYLENGYNQNH402GR (2031.88 Da; Fig. 3A, peptide 2a) the presence of V, I, H, and Y variants of CFH, respectively and CYFPYLENGYNQNY402GR (2057.88 Da; Fig. 3B, peptide (Table 1). For example, the presence of peaks 1148 and 2031 2b). Because all four peptides contain cysteine residues, they in the absence of peaks 1162 and 2057 corresponded with the were found to be predominantly modified with iodoacetamide VVHH CFH allotypic variant, whereas the presence of peaks (whose mass is included in these peptide masses). In addition, 1148, 1162, and 2031 and the absence of peak 2057 corre- small fractions of peptides 1a and 1b (Ͻ25%) were also found sponded to the VIHH variants. We also conducted a masked to be derivatized with oxygen at the methionine residue (not analysis for detection of unknown genotypes of CFH in nine shown). However, this oxidation did not significantly reduce human blood samples and found a 100% correlation between the main mass peaks of peptides 1a and 1b, which where used the peptide mass peak patterns described herein and the cor- for CFH isoform identification. responding patient’s CFH genotype.

FIGURE 4. MS spectra of plasma samples collected from individuals with genotypes VV62 (A), II62 (B), and VI62 (C).

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FIGURE 5. MS spectra of plasma samples genotyped as HH402 (A), YY402 (B), and HY402 (C).

DISCUSSION isoforms with 100% accuracy. We should stress that, although the MS/MS analysis was used to confirm the identity of each MS is a rapidly developing analytical tool for protein identifi- peptide, MS analysis alone was sufficient for reliable peptide cation in complex biological samples. The high resolution and identification. Therefore, a basic inexpensive mass spectrome- sensitivity of this method provides extensive sequence cover- ter capable of performing only peptide fingerprinting (MS age for proteins of interest, facilitates the analysis of posttrans- analysis of peptides without their fragmentation) could be used lational modifications, and allows identification of sequence for this assay. This makes it feasible for use in many clinical and variations that often have important physiological conse- analytical laboratories. quences. Recent studies have highlighted the utility of the Currently, there are several potentially important appli- MS-based approaches for detection of microheterogeneity and cations for this assay. First, it can be used to test nongeno- allotypic variations of plasma proteins including hemoglobin, typed human plasma samples that are available in large C-reactive protein, transferrin and Cu/Zn-superoxide dis- amounts from commercial sources and cannot be genotyped mutase.8,9 This established MS as a reliable alternative to DNA due to complete removal of cells and, therefore, DNA. These genotyping in identification of protein isoforms, which is par- plasma samples provide an excellent source from which ticularly useful when biological fluids (e.g., plasma or serum) each CFH variant could be purified for biochemical studies. available for the analysis do not contain DNA. Second, genotyping of CFH in blood of patients who un- We have developed a rapid and sensitive method for detec- dergo liver transplantation, unless performed on biopsy tion of the I62V and H402Y variants of CFH in human plasma specimens, may not accurately reflect the circulating CFH samples based on a combination of crude fractionation, gel protein variant, due to the relative contribution of secreted separation, and MS. This method requires only few microliters CFH synthesized in the nonhomologous transplanted organ. of plasma and is suitable for analyzing multiple samples. The In this case an MS-based plasma assay could be used to analysis of many plasma samples with known genotypes (both determine the distribution of the CFH variants in a trans- masked and unmasked) based on the presence or absence of plantation patient’s plasma. four characteristic peptides allowed us to identify all nine CFH A future application of an MS-based plasma assay for CFH, which would require further modification into a quantitative

TABLE 1. Correlation between CFH Genotypes and the Characteristic method, would be to use it to determine relative amounts of Mass Peaks CFH variants based on the relative intensity of the characteristic mass peaks. This kind of assay could be a valuable tool for Mass Peaks (Da) population-based studies for measurement of the distribution of different CFH variants in pooled serum samples from certain 1148 1162 2031 2057 cohorts of subjects and patients. Genotype Peptide 1a Peptide 1b Peptide 2a Peptide 2b

VVHH Yes No Yes No Acknowledgments VIHH Yes Yes Yes No The authors thank Lisa S. Hancox for coordinating the plasma acqui- IIHH No Yes Yes No sition from the University of Iowa. VVHY Yes No Yes Yes VIHY Yes Yes Yes Yes IIHY No Yes Yes Yes References VVYY Yes No No Yes VIYY Yes Yes No Yes 1. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymor- IIYY No Yes No Yes phism in age-related macular degeneration. Science. 2005;308: 385–389.

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2. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H 6. Hakobyan S, Harris CL, Tortajada A, et al. Measurement of factor H variant increases the risk of age-related macular degeneration. variants in plasma using variant-specific monoclonal antibodies: Science. 2005;308:419–421. application to assessing risk of age-related macular degeneration. 3. Edwards AO, Ritter R 3rd, Abel KJ, Manning A, Panhuysen C, Farrer Invest Ophthalmol Vis Sci. 2008;49:1983–1990. LA. Complement factor H polymorphism and age-related macular 7. Fenaille F, Le Mignon M, Groseil C, et al. Site-specific N-glycan degeneration. Science. 2005;308:421–424. characterization of human complement factor H. Glycobiology. 4. Hageman GS, Anderson DH, Johnson LV, et al. A common haplo- 2007;17:932–944. type in the complement regulatory gene factor H (HF1/CFH) 8. Nedelkov D, Kiernan UA, Niederkofler EE, Tubbs KA, Nelson RW. predisposes individuals to age-related macular degeneration. Proc Investigating diversity in human plasma proteins. Proc Natl Acad Natl Acad SciUSA.2005;102:7227–7232. SciUSA.2005;102:10852–10857. 5. Abrera-Abeleda MA, Nishimura C, Smith JL, et al. Variations in the 9. Shimizu A, Nakanishi T, Miyazaki A. Detection and character- complement regulatory genes factor H (CFH) and factor H related 5 ization of variant and modified structures of proteins in blood (CFHR5) are associated with membranoproliferative glomerulone- and tissues by mass spectrometry. Mass Spectrom Rev. 2006; phritis type II (dense deposit disease). J Med Genet. 2006;43:582–589. 25:686–712.

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