ARTICLE pubs.acs.org/ac
Utility of Mass Spectrometry in the Diagnosis of Prion Diseases Christopher J. Silva,*,† Bruce C. Onisko,‡ Irina Dynin,† Melissa L. Erickson,† Jesus R. Requena,§ and John Mark Carter† †
Western Regional Research Center, United States Department of Agriculture, Albany, California 94710, United States OniPro Biosciences, Kensington, California 94707, United States § Prion Research Unit, Department of Medicine, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain ‡
bS Supporting Information ABSTRACT: We developed a sensitive mass spectrometrybased method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol. Nonanalyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice 5 weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or four mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the nonobex/nonanterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.
A
prion is the etiological agent of transmissible spongiform encephalopathies (TSEs). Prion diseases have long asymptomatic incubation periods followed by a relatively short symptomatic period that ends in the death of the host. A prion (PrPSc) is able to induce the normal cellular prion protein (PrPC) to undergo a conformational change that converts it into PrPSc and thereby propagate an infection.1 Prion diseases can infect a wide variety of agriculturally important animals such as sheep and cows.2,3 Wild and farmed deer, elk, moose, mink, and zoo animals have succumbed to TSE diseases.4-6 Creutzfeldt-Jakob disease (CJD), Gerstmann-Str€aussler-Scheinker disease, kuru, and fatal familial insomnia are the known human prion diseases. Prion diseases can be inherited, sporadic, or transmitted. Extensive structural analysis has shown that PrPC and PrPSc possess identical covalent structures. The primary amino acid sequence of PrPC and PrPSc is identical. They have identical posttranslational modifications, including a single disulfide bond, covalently attached sugar antennae, and a single glycophosphatidylinositol (GPI) anchor.7 The sugar antennae are covalently bound to asparagine residues.7 These sugar antennae are highly varied, and while the composition of the sugar antennae is variable, it varies similarly in both PrPC and PrPSc. The chemical structure of the GPI anchor also varies similarly in both PrPC and r 2011 American Chemical Society
PrPSc. There is no covalent difference between PrPC and PrPSc, thus the structural differences between PrPC and PrPSc are entirely conformational. Although there are no covalent differences between PrPC and PrPSc, they have significantly different physicochemical properties. PrPC is monomeric and soluble in nondenaturing detergents. In contrast, PrPSc is a multimer of PrPC molecules that have been refolded into a non-native conformation. Digestion of PrPSc with proteinase K yields a characteristic N-terminally truncated protein referred to as PrP 27-30. A similar digestion of PrPC would completely degrade the protein. The PrPSc multimer can be separated from the PrPC monomer by ultracentrifugation.8-16 Ultracentrifugation yields pellets containing >90% of the infectivity present in the samples.16 Detection of prions is challenging. An infectious dose is estimated to be approximately one million molecules.17,18 Animal bioassay, using rodent models, is the most sensitive prion detection method, but it requires a large number of laboratory animals and takes a long time, so it is not a practical assay Received: September 23, 2010 Accepted: January 10, 2011 Published: February 02, 2011 1609
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Analytical Chemistry method.19 The limit of detection reported for Western blot is approximately 10-20 pmol.20-22 Enzyme-linked immunosorbent assays (ELISAs) are more sensitive and have been reported to detect 2 pmol.23-25 The conformation dependent immunoassay (CDI) is even more sensitive (0.1 pmol).26 We developed a method of mass spectrometry based quantitation of PrP 27-30 in hamsters infected with the 263K strain of hamster-adapted scrapie.27,28 The limit of detection for this method is considerably lower than any of the previously described detection methods (20-30 amol). This approach exploits the mass spectrometry-based multiple reaction monitoring (MRM) method of quantitation. In principle this MRM method can be used to quantitate prions from species other than hamsters. Additionally the MRM method can be used to detect the presence of other PrP 27-30 derived trypsin fragments. This approach can be used to both quantitate and confirm the presence of PrP 27-30. In this way mass spectrometry can be used to diagnose prion disease. We wish to report our development of a method of quantitating prions in nonhamster species and the use of the MRM method to detect other characteristic PrP 27-30 trypsin fragments. This combined approach will allow for a general mass spectrometry based method of diagnosing prion diseases.
’ EXPERIMENTAL PROCEDURES Chemicals. The synthetic peptides described in this work were synthesized by Anaspec (Fremont, CA). The chromatographic mobility of the isotopically labeled internal standards and their corresponding peptides is identical. HPLC grade water was purchased from Burdick and Jackson (Muskegon, MI). Acetonitrile, HPLC grade, was from Fisher Scientific (Fairlawn, NJ). Trypsin (porcine, sequencing grade, modified) was purchased from Promega (Madison, WI). All other reagents were from Sigma-Aldrich (St. Louis, MO). Animal Handling and Sample Preparation. LVG Syrian golden hamsters and Swiss CD-1 mice were obtained from commercial sources (Charles River Laboratories, Wilmington, MA). Uninfected cow and sheep brains were obtained from commercial slaughterhouses (Dixon, CA). Brains from mule deer were obtained from hunter harvested animals from a CWD-free area of the United States (Florida). The drowsy strain was obtained from InPro Biotechnology (South San Francisco, CA). The 139H, 22AH, and 22CH strains of hamster-adapted scrapie were gifts from Richard I. Carp. The hamster strains were passaged once through LVG Syrian golden hamsters (Charles River Laboratories, Wilmington, MA 01887). The Me7 and RML strains of mouse-adapted scrapie were obtained from InPro Biotechnology (South San Francisco, CA 94080). The Me7-298 and 79A strains of mouse-adapted scrapie were obtained from the TSE Resource Centre (IAH, Compton, U.K.). The sheep brains were harvested from naturally infected animals that were humanely euthanized in the terminal stages of the disease. The scrapie strain infecting these animals was not the “atypical” or Nor98 strain. Three LVG hamsters or Swiss CD-1 mice were inoculated (ic) with 50 or 30 μL of a 10% brain homogenate, respectively, of the hamster or mouse-adapted prion strains. The homogenates were prepared from the brain of a terminal-stage animal. After 5 weeks of incubation, the 12 hamsters and mice were euthanized. The brain was quickly removed from the skull of the animal and immediately frozen at -80 °C.
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PrPSc or control material was isolated according to the methods of Bolton et al. with some minor modifications.16 The isolated PrPSc or control material was prepared for mass spectrometric analysis by previously described methods.28,29 Production of Recombinant PrP. Recombinant PrP was obtained from plasmids expressing the protein sequence. The sheep plasmid (pTrcHis-TOPO; Invitrogen; Carlsbad, CA) was a gift from J€urgen Richt (USDA; currently at Kansas State University). The plasmids (pET-11a; Novagen; Darmstadt, Germany) expressing the hamster and mouse sequences were a gift from Carsten Korth. The plasmids were cloned into BL21 cells (Novagen; Darmstadt, Germany). The molecular weight of each protein was verified by mass spectrometry. All of the proteins contained an N-terminal methionine.30 The recombinant proteins were isolated using standard molecular biology techniques and published methods.31,32 Mass Spectrometry. The instrument response was optimized by a previously described method.28 The mass spectrometer was operated in multiple reaction monitoring (MRM) mode, alternating between detection of three peptides and the appropriate internal standards. The mass settings for the peptides included the following: VVEQMCTTQYQK (iodoacetamide derivative; precursor ion m/z of 757.8, product ion of m/z 171.1 (a2 ion)), VVEQM(SO)CTTQYQK (iodoacetamide derivative; precursor ion m/z of 765.8, product ion of m/z 171.1 (a2 ion)), GENFTETDIK (precursor ion m/z of 577.3, product ion of m/z 706.4 (y6 ion)), and ESQAYYDGR (precursor ion m/z of 544.7, product ion of m/z 510.4 (y4 ion) or m/z 673.4 (y5 ion)), VVEQMCVTQYQK (iodoacetamide derivative; precursor ion m/z of 756.8, product ion of m/z 171.2 (a2 ion)), VVEQM(SO)CVTQYQK (iodoacetamide derivative; precursor ion m/z of 764.8, product ion of m/z 171.2 (a2 ion)), GENFTETDVK (precursor ion m/z of 570.3, product ion of m/z 692.3 (y6 ion)), VVEQMCITQYQR (iodoacetamide derivative; precursor ion m/z of 778.1, product ion of m/z 171.1 (a2 ion)), VVEQM(SO)CITQYQR (iodoacetamide derivative; precursor ion m/z of 786.1, product ion of m/z 171.1 (a2 ion)), VVEQMCITQYER (iodoacetamide derivative; precursor ion m/z of 778.6, product ion of m/z 171.1 (a2 ion)) and its respective sulfoxide VVEQM(SO)CITQYER (iodoacetamide derivative; precursor ion m/z of 786.6, product ion of m/z 171.1 (a2 ion)), [13C5,15N]VVEQMCTTQYQK (iodoacetamide derivative; precursor ion m/z of 760.8, product ion of m/z 177.1 (a2 ion)), [13C5,15N]VVEQMCVTQYQK (iodoacetamide derivative; precursor ion m/z of 759.9, product ion of m/z 177.2 (a2 ion)), [13C5,15N]VVEQMCITQYQR (iodoacetamide derivative; precursor ion m/z of 781.1, product ion of m/z 177.1 (a2 ion)), [13C5,15N]GENFTETDIK (precursor ion m/z of 580.8, product ion of m/z 713.4 (y6 ion)), and [13C9,15N]-GENFTETDVK (precursor ion m/z of 575.3, product ion of m/z 702.4 (y6 ion)). The optimal collision energies for the peptides were determined to be VVEQMCITQYER and VVEQM(SO)CITQYER (iodoacetamide derivative, 48 V), VVEQMCTTQYQK and VVEQM(SO)CTTQYQK (iodoacetamide derivative, 47 V), VVEQMCITQYQR and VVEQM(SO)CITQYQR (iodoacetamide derivative, 50 V), GENFTETDIK (28 V), GENFTETDVK (28 V), ESQAYYQR (30 V), and ESQAYYDGR (33 V)). Quantitation was done with the Intelliquan quantitation algorithm using Analyst 1.4.1 software. Safety Considerations. Acetonitrile is hazardous and was manipulated in a dedicated chemical safety hood. Prions are infectious, so all prion-containing samples were manipulated in a 1610
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Analytical Chemistry dedicated biosafety level 2 (BSL-2) laboratory (certified and inspected by the Animal and Plant Health Inspection Service (APHIS) of the USDA (www.aphis.usda.gov/permits/)) using procedures outlined in the fifth edition of the CDC’s biosafety manual, Biosafety in Microbiological and Biomedical Laboratories (www.cdc.gov/biosafety/publications/bmbl5). The infectious material was inactivated before removal from the dedicated BSL-2 laboratory by the addition of a sufficient volume of 8 M guanidine hydrochloride to make a 6 M solution that was thoroughly mixed and allowed to stand for at least 24 h at room temperature. The solution of inactivated prions was transferred to clean fresh tubes and removed from the BSL-2 laboratory.
’ RESULTS Mouse, sheep, and hamster recombinant PrP and PrP 27-30 were selected as representatives of the broader range of mammalian prions. The tryptic peptides selected for this study were based on the results previously obtained for hamsters.28 The tryptic peptides for the typical sheep, deer, and cow PrP (VVEQMCITQYQR, GENFTETDIK, and ESQAYYQR) are identical. Two of the peptides derived from mouse (VVEQMCVTQYQK, GENFTETDVK, and ESQAYYDGR) and hamsters (VVEQMCTTQYQK, GENFTETDVK, and ESQAYYDGR) are identical and one is different. The typical human sequence has one distinct tryptic peptide (VVEQMCITQYER), one in common with mouse (GENFTETDVK), and one common to sheep, cow, and deer (ESQAYYQR). This set of peptides can be used to analyze PrPSc in a wide variety of animal models and agriculturally important animals. The instrument response was optimized for each of the 10 selected peptides.28 The signal for each analyte peptide, its oxidized analogue and four other peptides (VVEQMCITQYER, VVEQMCITQYQR, VVEQMCVTQYQK, VVEQM(SO)CITQYER, VVEQM(SO)CITQYQR, VVEQM(SO)CVTQYQK, GENFTETDIK, GENFTETDVK, ESQAYYDGR, or ESQAYYQR) was maximized by adjusting the source parameters and the Q2 offset voltage (“collision energy”) to yield optimal fragmentation (Table S-1 in the Supporting Information). The listed ions and those described in previous work were used throughout this manuscript.28,29 In addition to the previously described hamster peptide (VVEQMCTTQYQK), three related peptides (VVEQMCITQYQR, VVEQMCVTQYQK, and VVEQMCITQYER) were determined to be suitable for quantitation.27-29 The asparagine (N) residue contained in the peptide GENFTETDIK or GENFTETDVK is covalently bound to sugar antennae. The extent of this glycosylation is variable, making these peptides unsuitable for quantitation. The peptides ESQAYYQR and ESQAYYDGR are located in a region of the protein that is cleaved by an uncharacterized protease.33 Although these four peptides are unsuitable for quantitation, they can be used to confirm the presence of PrP 27-30. The limit of detection (LOD) and limit of quantitation (LOQ) were determined for the three analyte peptides, VVEQMCITQYQR, VVEQMCVTQYQK, and VVEQMCITQYER. The LOQ and LOD for the hamster (VVEQMCTTQYQK)28 and mouse peptides (VVEQMCVTQYQK) were similar and lower than that for sheep, cow, and deer (VVEQMCITQYQR) and human (VVEQMCITQYER). The LOQ and LOD of each of these peptides were below 100 amol, as were those reported for the hamster peptide.28
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A series of solutions containing known but varying concentrations of individual analyte peptides or oxidized homologues and a fixed amount of the appropriate isotopically labeled internal standard were prepared. After triple quadrupole mass spectrometric analysis, the area ratio of the integrated signal from the peptide to that of the isotopically labeled internal standard was used to prepare a calibration curve (Figure S-1 in the Supporting Information). The correlation coefficient for these quadratic curves is 0.99. Furthermore, the coefficient of variation (CV) for these area ratios is small (0.9-3.8%). The CV for the analyte retention time was low (∼2.0%). These results indicate that this method provides an accurate and reproducible way of quantitating the analyte peptide and its corresponding sulfoxide. This allows for an accurate quantitation of PrP 27-30. The analyte peptides and their respective sulfoxide analogues have different chromatographic retention times. Under the chromatographic conditions we employed, the sulfoxide diastereoisomers have identical chromatographic retention times. They separate from the unoxidized peptides by baseline separation. The isotopically labeled internal standards and their respective nonlabeled analogues have identical chromatographic properties. The two forms differ solely in terms of their respective molecular masses. This allows them to be used to determine the relative chromatographic retention of a given peptide fragment. The use of internal standards permits the identification of a peptide by its chromatographic properties, distinct precursor m/z ratio, and the m/z ratio of its characteristic fragments. The amount of oxidized analyte peptide found in these samples was similar to that previously reported.29 The MRM method permits the rapid sequential detection of signals from a number of peptides. Such signals can be a single signal from one peptide or multiple signals from the same peptide. Although the four nonanalyte peptides (GENFTETDIK, GENFTETDVK, ESQAYYQR, and ESQAYYDGR) are not suitable for quantitation, they can be used to confirm the presence of recombinant PrP or PrP 27-30. In this way the MRM method can be used, in the same experiment, to quantitate PrP 27-30 based on the signal intensity of the analyte peptide and to confirm the presence of PrP 27-30 based on the presence of a signal from the two appropriate nonanalyte peptides (e.g., for hamsters GENFTETDIK and ESQAYYDGR). Samples of hamster, mouse, or sheep recombinant prion protein were subjected to MRM analysis. The instrument was set to detect the appropriate peptides and internal standards from each of the three species. The signal intensities were recorded and plotted. Figures 1A, 2A, and 3A show the results for the hamster, sheep, and mouse experiments, respectively. In the recombinant samples, the peptide that generates the most intense signal for mouse or hamster is GENFTETDIK. For sheep the most intense signal is from the homologous peptide, GENFTETDVK. Unlike the other eight peptides, ESQAYYQR (sheep) and ESQAYYDGR (mouse or hamster) produce two fragment ions (y4 and y5 ions) of comparable intensity. The least intense signal comes from the analyte peptide. The relative intensity of the signals from these peptides is similar for recombinant protein from sheep, mouse, and hamster. Samples of brain tissue from prion infected hamsters, mice, or sheep were subjected to the same MRM analysis. After isolating PrP 27-30 from these three species and subjecting it to trypsin digestion, the samples were subjected to the mass spectrometric analysis that was appropriate for each species. The signal intensities were recorded and plotted. Figures 1B, 2B, and 3B 1611
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Figure 1. Comparison of the intensity of the MRM signals from the peptides VVEQMCTTQYQK (i), GENFTETDIK (ii), and ESQAYYDGR (iii and iv) derived from the trypsin digest of recombinant hamster PrP (A) or hamster (263K strain) PrP 27-30 (B).
Figure 3. Comparison of the intensity of the MRM signals from the peptides VVEQMCVTQYQK (i), GENFTETDVK (ii), and ESQAYYDGR (iii and iv) derived from the trypsin digest of recombinant mouse PrP (A) or mouse (RML strain) PrP 27-30 (B).
Figure 2. Comparison of the intensity of the MRM signals from the peptides VVEQMCITQYQR (i), GENFTETDIK (ii), and ESQAYYQR (iii and iv) derived from the trypsin digest of recombinant sheep PrP (A) or sheep PrP 27-30 (B).
chromatographic retention time as the selected peptide. It would have to possess a similar precursor m/z ratio (0.5 Da and generate, in the N2 collision cell, a product fragment with a similar m/z ( 0.5 Da. Although highly unlikely, interference from a molecule possessing such a combination of these three diverse properties cannot be excluded. In order to test for such interference, we removed the brains from uninfected animals and subjected them to mass spectrometric analysis. Brains from uninfected sheep, deer, cow, mice, and hamsters were removed and subjected to the prion isolation procedure. The resulting pellets were subjected to mass spectrometric analysis using the internal standards. A careful examination of the resulting chromatograms revealed that there was no signal greater than noise for any of the 10 peptides which would confound this method of analysis (Figure S-2 in the Supporting Information). These results were similar to those previously reported for analyte peptides and the oxidized form of the analyte peptide in hamsters.27,29 We conclude, therefore, that there are no interfering molecules present in a healthy brain and that the signals we observe from infected brains result from PrP 27-30. A given host can be infected by more than one kind of prion. These different prions have distinct phenotypes and are referred to as strains. Two strains of mouse-adapted sheep scrapie (Me7and Me7-298), two strains of mouse-adapted goat scrapie (79A and RML), two strains of hamster-adapted sheep scrapie (22AH and 22CH), one strain of hamster-adapted goat scrapie (139H), and one strain of hamster-adapted transmissible mink encephalopathy (drowsy) were used. The incubation periods of these eight rodent-adapted prion strains are in excess of 130 days. The typical incubation period of the mouse-adapted strains ranged from 152 to 158 days (Me7-298, 155 days; Me7, 152 days; 79A, 158 days; RML 157 days). The hamster-adapted prions strains had incubation periods between 129 and 176 days (22AH, 158 days; 22CH, 129 days; drowsy, 168 days; 139H, 176 days). Each strain was inoculated ic into either three hamsters (hamster-adapted prions) or mice (mouse-adapted prions). After 5 weeks, the infected but asymptomatic animals were euthanized and their brains removed. After removal, the brains
show the results for the hamster, sheep, and mouse experiments, respectively. In these samples the intensity of signal from the GENFTETDIK or GENFTETDVK peptide is significantly reduced due to the glycosylation present in PrP 27-30. The relative signal intensity from either the ESQAYYQR (sheep) or ESQAYYDGR (mouse or hamster) varies compared to that of the analyte peptide. This is due to the modification of the C-terminal portion of the protein.33 Unlike the recombinant material, the relative intensity from these peptides varies considerably from one species to another. There is a possibility that some other molecules might produce signals that would interfere with this method. The internal standards were of sufficiently high isotopic purity that no signal was observed for the analyte peptide when only the isotopically labeled internal standard was subjected to mass spectrometric analysis. In order for some other molecule to interfere with this analysis, it would have to possess a similar
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Figure 4. Comparison of the intensity of the MRM signals from peptides present in trypsin digests of hamster (139H, 5 weeks post inoculation, 1 fmol injected) (A), mouse (Me7, 5 weeks post inoculation, 1.2 fmol injected) (B), or sheep (3 fmol injected) (C) PrP 27-30. The graphs are offset for clarity. Graph i corresponds to the signals from to the peptides VVEQMCTTQYQK (A, hamster), VVEQMCVTQYQK (B, mouse), or VVEQMCITQYQR (C, sheep). Graph ii corresponds to signal from the peptides GENFTETDIK (hamster and sheep) and GENFTETDVK (B, mouse). Graphs iii and iv correspond to the signals from the peptides ESQAYYDGR (A, hamster and B, mouse) and ESQAYYQR (C, sheep).
were processed and subjected to mass spectrometric analysis. Each animal had detectable amounts of PrP 27-30, as measured by the sum of the analyte peptide and the oxidized form of the analyte peptide. The mice infected with the Me7 and Me7-298 strains had the highest amount of mouse PrP 27-30 (Me7, 2 102 ( 5 101 fmol; Me7-298, 1 102 ( 8 101 fmol). Mice infected with the 79A strain had less (4 101 ( 4 fmol), and those infected with the RML strain had the lowest amount among mice (3 101 ( 4 fmol). Hamsters infected with the 22CH and 22AH strains had the highest amount of PrP 27-30 present in their brains, 1 103 ( 6 102 and 2 102 ( 7 101 fmol, respectively. Hamsters infected with the drowsy and 139H strain had lower amounts of PrP 27-30, 5 101 ( 2 101 and 7 101 ( 3 101 fmol, respectively. This variation seems to be dependent upon the strain phenotype (incubation period and source animal species). One scrapie infected sheep brain was obtained from an animal in the terminal stages of the disease. It was sliced into three sections: the obex, the anterior portion of the cerebrum, and the remaining nonobex/nonanterior portion of the cerebrum. The prions from each portion were isolated and subjected to mass spectrometric analysis. The concentration of PrP 27-30 in the anterior portion of the cerebrum was determined to be 3 102 ( 1 102 fmol per gram of tissue. The quantity of PrP 27-30 present in a brain homogenate made from the brain tissue not including the obex or the anterior portion of the cerebrum was determined to be 4.6 104 ( 2 103 fmol per gram. The obex contained 5.9 104 ( 7 103 fmol of PrP 27-30 per gram. The MRM intensities were recorded and plotted for the 139H strain of hamster-adapted scrapie, the Me7 strain of mouseadapted scrapie, and the nonobex portion of the scrapie-infected sheep brain. These results are shown in Figure 4. The 139H sample represents the signal from approximately 1 fmol of analyte peptide (Figure 4A). The sample from the Me7 strain of mouse adapted scrapie contains approximately 1 fmol of analyte peptide (Figure 4B). The sheep scrapie sample represents the signal from approximately 3 fmol of analyte peptide (Figure 4C). It is clear from these figures that although the other peptides, GENFTETDIK (hamster, sheep), GENFTETDVK
(mouse), ESQAYYQR (sheep), and ESQAYYDGR (mouse, hamster), may not be suitable for quantitation, they are certainly suitable for confirmation. Our MRM approach allows for both the quantitation and confirmation of prions.
’ DISCUSSION This mass spectrometry-based method uses chromatographic retention time and characteristic MRM fragments to quantify and identify characteristic trypsin fragments from PrP 27-30 from a variety of prion infected species. The LOD and LOQ values for the analyte peptides from sheep, cow, deer, mouse, hamster, and human PrP are below 100 amol. In addition, other characteristic trypsin fragments, while not suitable for quantitation, can be used to confirm the presence of PrP 27-30 from an infected animal. This approach was successfully used to detect prions in various portions of the brains of sheep. It was successfully used to quantify the amount of PrP 27-30 present in the brains of preclinical experimental rodents infected with several prion strains. This approach can be used to detect and quantify a variety of prions in a number of species. The method allows one to detect PrP 27-30 well before the appearance of pathological lesions. Mice infected with the Me7 strain of mouse-adapted scrapie show signs of pathology only after 60 days.34,35 This is similar to that observed in the 79A strain of mouse-adapted scrapie.36 The earliest evidence for pathology observed in the drowsy strain of hamster-adapted mink encephalopathy is 11 weeks.37 The 22CH strain showed evidence of pathology at 60 days.38 Since PrP 27-30 was detectable by 35 days post inoculation for all of these strains by mass spectrometry, this approach permits the detection of prions before there is any evidence of pathological damage. Before pathological changes occur, the accumulation of PrPSc can be observed in experimental animals by a variety of methods. In mice infected with the RML strain of PrPSc, accumulation was detectable 49 days post (ic) inoculation by histoblotting.39 By Western blot, PrP 27-30 was detectable after 70 days.39 We were able to detect PrP 27-30 in mice infected with the RML strain after just 35 days. The accumulation of PrPSc was barely 1613
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Analytical Chemistry detectable by histoblot in the 139H strain 40 days post inoculation.40 The signal observed from the mass spectrometric analysis (Figure 4A) was of sufficient intensity that, based on a doubling time of 1 week,27 PrP 27-30 should be detectable in 139H infected hamsters after only 21 days. Using a highly sensitive form of histoblotting, researchers were able to detect the accumulation of PrP 27-30 in mice infected with the Me7 strain of mouse-adapted goat scrapie after 30 days.41 The signal intensity observed from our analysis (Figure 4B) after 35 days suggests that PrP 27-30 would be detectable after 21 days. Our mass spectrometry-based approach is more sensitive than the current methods of detecting prions prior to observable pathology. The obex is used for the diagnosis of prion diseases in sheep and other agriculturally important animals. In most sheep infected with scrapie (typical), the concentration of prions is highest in this area, so it offers the best chance of detecting the disease. In a new strain of scrapie, Nor98 or atypical scrapie cases, prions are found in highest concentration in the cortex of the cerebellum and the cerebrum, not in the obex.42 Our mass spectrometry-based approach permits the detection of prions in regions of the brain other than the obex. This means that it can be used to detect prions in nonobex portions of the brain or in atypical cases. The mass spectrometry-based approach does not require intact tissue for analysis. It can be used to detect prions from degraded tissue, where the lack of tissue integrity makes a pathological examination impossible. Polymorphisms in the PrPC of various animals can influence a mass spectrometry-based analysis. Synonymous polymorphisms in the DNA sequence would not interfere with this analysis since they encode the same amino acid. If a lysine (K) or arginine (R) was replaced with another amino acid (not R or K), then trypsin cleavage would not occur and the expected peptide would not be present for analysis. If the amino acid in a selected peptide was changed, then the molecular weight, chromatographic properties, and characteristic fragmentation would change and the peptide would no longer be detectable. In order to confound this analysis, a PrPC polymorphism would have to occur in an amino acid in the 37-39 amino acids (depending on the species) nearest the C-terminus of PrPC. There are a number of known PrPC polymorphisms in experimental and agriculturally important animals, but most are not present in the 37-39 amino acids near the C-terminal end of the protein. Hamsters have no known PrPC polymorphisms, so there is no interference with this mass spectrometrybased method of analysis. Mice have two polymorphisms (L108F and T189 V), but neither of these would interfere with this analysis. Sheep have at least 20 polymorphisms.43 Of the 20 sheep polymorphisms only one of these, R211Q, would impact this approach. Homozygous R211Q sheep would no longer produce the analyte peptide (VVEQMCITQYQR) after trypsin digestion. Such animals would produce the other two peptides (GENFTETDIK and ESQAYYQR), which could be used to verify the presence of prions but not to quantitate them. Cows have at least four polymorphisms.43,44 The E211K polymorphism would interfere with this analysis, since the peptide GENFTETDIK would no longer be present. The analyte peptide (VVEQMCITQYQR) and the other confirming peptide (ESQAYYQR) would be unchanged by this polymorphism. Deer have at least five polymorphisms, but none of these polymorphisms would influence this analysis.43
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A much larger number of polymorphisms are found in the human PrPC protein.45 Most of these polymorphisms are associated with familial prion diseases. The known polymorphisms E196K, F198S, E200K, D202N, and V203I would prevent the detection of one of the peptides (GENFTETDIK) but not the analyte peptide (VVEQMCITQYER) or the other peptide (ESQAYYQR). The polymorphisms R208H, V210I, E211Q, Q212P, Q217R, and E219K would prevent the detection of the analyte peptide (VVEQMCITQYER) but not the other two peptides (GENFTETDIK and ESQAYYQR). Only the Y145Stop and Q160Stop polymorphisms would be undetectable, since the peptide is truncated at amino acid 145 or 160, respectively. Although the specific examples used in this study are limited to mouse, hamster, and sheep samples, this approach is a general method that can be applied to a variety of species. It has been successfully used on a variety of prion strains from mice and hamsters. It allows for the direct detection of pathogens (prions) that are isolated by ultracentrifugation. It even permits their detection before there is evidence of pathology and is more sensitive than the most sensitive antibody-based approach.27 In principle, it can be used to detect the presence of prions regardless of the known polymorphisms. In addition, this approach can be used to both detect and quantitate the vast majority of prions, based on the known prion polymorphisms. Furthermore, it allows for the confirmation of the presence of prions by detecting the signals from other peptides derived from the trypsin digestion of PrP 27-30. In this way it can be used to both quantitate and to confirm the presence of prions in the attomole range.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Address: USDA, ARS, 800 Buchanan Street, Albany, CA 94706. Phone: (510) 559-6135. Fax: (510) 559-5758. E-mail:
[email protected].
’ ACKNOWLEDGMENT Supported by Grant LHSB-CT-2006-019090 from the EU and Grant BFU2006-04588/BMC from the Spanish Ministry of Science and Education to J.R.R. ’ REFERENCES (1) Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363– 13383. (2) Detwiler, L. A.; Baylis, M. Rev. Sci. Tech. 2003, 22, 121–143. (3) Harman, J. L.; Silva, C. J. J. Am. Vet. Med. Assoc. 2009, 234, 59–72. (4) Kirkwood, J. K.; Cunningham, A. A. Vet. Rec. 1994, 135, 296– 303. (5) Marsh, R. F.; Hadlow, W. J. Rev. Sci. Tech. 1992, 11, 539–550. (6) Williams, E. S. Vet. Pathol. 2005, 42, 530–549. (7) Stahl, N.; Baldwin, M.; Teplow, D. B.; Hood, L. E.; Beavis, R.; Chait, B.; Gibson, B. W.; Burlingame, A. L.; Prusiner, S. B. In Prion Diseases of Humans and Animals; Prusiner, S. B., Collinge, J., Powell, J., Anderton, B., Eds.; Ellis Horwood: New York, 1992; pp 361-379. 1614
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