Mass spectrometric analysis of prion proteins

MASS SPECTROMETRIC ANALYSIS OF PRION PROTEINS BY MICHAEL A. BALDWIN Mass Spectometry Facility, University of California, San Francisco, California 94143

I. II. III. IV. V. VI.

VII. VIII. IX. X. XI.

Modern Mass Spectrometric Techniques for Protein Characterization . . . . Identification and Preliminary Analysis of PrP . . . . . . . . . . . . . . . . . . . . . . . . Confirmation of the PrPSc Amino Acid Sequence. . . . . . . . . . . . . . . . . . . . . . Non-PrP Peptides in Prion Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N -linked Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of the GPI Anchor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. ESIMS of the C-terminal Peptide-GPI. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Branching Patterns in the GPI Glycan by MS/MS . . . . . . . . . . . . . . . . . . C. Unanswered Questions Concerning the GPI . . . . . . . . . . . . . . . . . . . . . . Analysis of Intact PrP by MALDIMS and ESIMS . . . . . . . . . . . . . . . . . . . . . . . Processing of Chicken PrP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recombinant PrP and Synthetic Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. PrP Modifications Due to Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Copper Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accessory Molecules in Scrapie Prions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 30 31 37 37 39 41 42 43 45 49 49 50 50 51 51 52

1. MODERN MASS SPECTROMETRIC TECHNIQUES FOR PROTEIN CHARACTERIZATION As a result of developments in the methods for desorption and ionization of polar and labile materials from condensed phase, mass spectrometry has become a powerful tool for biochemical analysis. In the 1980s the new methods of fast atom bombardment mass spectrometry (FABMS) (1) and liquid secondary ionization mass spectrometry (LSIMS) (2) allowed the analysis and sequencing of peptides without derivatization, and the detection and identification of posttranslational modifications (3). These methods enabled the routine, rapid, and accurate mass analysis of peptides isolated from proteolytic or chemical digestions, complementing longer established chemical techniques such as amino acid analysis and Edman N-terminal sequencing. More recently these methods were supplanted by electrospray ionization (ESI) (4, 5) and matrix assisted laser desorption/ionization (MALDI) (6–9). In addition to being applicable to peptides, these latter techniques have proved to be capable of ionizing large intact proteins and providing highly accurate molecular masses, in favorable cases with 29 ADVANCES IN PROTEIN CHEMISTRY, Vol. 57

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parts-per-million accuracy and with subpicomole sensitivity. They are also applicable to the analysis of nucleic acids including DNA and RNA, oligosaccharides, and lipids (10). Much of the success of these so-called “soft ionization” methods results from the low levels of vibronic excitation imparted to the molecules during the ionization process. Thus molecular ions are less likely to break down and they are mostly preserved intact, even for labile and potentially unstable compounds. Structural information can be obtained by collision-induced dissociation (CID) of these preponderant molecular ions in a tandem mass spectrometer, which can lead directly to the sequence of amino acids in a peptide or the sequence and branching of sugar units in an oligosaccharide. This technique is also referred to as mass spectrometry/mass spectrometry (MS/MS). Today mass spectrometry is capable of determining the amino acid sequence and posttranslational modifications of virtually any protein that can be purified in sufficient quantity. II. IDENTIFICATION AND PRELIMINARY ANALYSIS OF PrP After PrP was first cloned in the mid-1980s (11), it was soon established that PrPSc and PrPC are both encoded by the same cellular gene and that PrPSc is derived from PrPC in a posttranslational event (12, 13). Despite dramatic differences in the physical properties of PrPC and PrPSc, biochemical analysis failed to identify any chemical differences. Both forms were shown to contain a single disulfide bond (14). They are modified with N-linked oligosaccharides that cause intact PrP to give three separate bands by SDS-PAGE at 33–35 kDa and that can be removed with peptide N-glycosidase F (PNGase F) (15, 16). Both forms bind to cellular membranes via a glycosyl phosphatidylinositol (GPI) group, the lipids of which can be removed by phosphatidylinositol phospholipase C (PIPLC) (17). A series of articles published from the University of California, San Francisco, between 1990 and 1993 described the use of mass spectrometry to address questions that might explain the pathological properties of PrPSc. Do processes such as RNA-editing cause changes in the amino acid sequence? Are there differences in posttranslational modifications of PrPC and PrPSc? Do any identified modifications possess unusual features that could explain the pathogenic nature of PrPSc?

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Is the protein homogeneous or could a subpopulation be responsible for the unusual properties? It is noteworthy that although mass spectrometry is now widely used for the identification and characterization of proteins, it is quite unusual to carry out a complete analysis in which every amino acid of a protein is probed. Measuring the molecular weights of a subset of peptides from a proteolytic digest or determining a region of amino acid sequence is normally sufficient to identify a protein present in a database. For previously unknown proteins or those for which no corresponding genetic sequence is available, some amino acid sequence will normally allow cloning and expression of recombinant products. If such a protein has the “correct molecular weight,” it is generally assumed to be the target protein, particularly if it has the same activity, although a protein expressed in bacteria is likely to lack the posttranslational modifications of the natural form. In the case of PrP, such an approach was not adequate to answer the questions posed previously. At the time there were well-established methods for purifying proteolytically truncated PrPSc from the brains of hamsters artificially infected with scrapie. PrPSc accumulates during disease and its insolubility and unusual resistance to proteolysis allow a truncated core (PrP 27–30) to be isolated after treatment with proteinase-K (18–20). This retains infectivity, despite the loss of up to 67 amino acids from the N-terminus. Much of the analysis was carried out using this form, although the full-length protein was also isolated in lower yield for analysis of the N-terminal region. Both forms required the presence of detergent for solubilization, and they were rendered more soluble by chaotropic agents such as 6 M guanidine hydrochloride. Although guanidine denatured the protein and destroyed the infectivity, it did not diminish the effectiveness of mass spectrometric determination of the primary structure. Before analysis the disulfide bond was reduced with dithiothreitol and carboxymethylated with iodoacetic acid. The GPI lipids were incompatible with reversed phase high-performance liquid chromatography (HPLC), consequently they were removed by digestion with PIPLC. PrPC was more difficult to isolate and at that time was only available in very small quantities (21). III. CONFIRMATION OF THE PrPSC AMINO ACID SEQUENCE The Syrian hamster PrP gene sequence was already known from partial purification of PrP 27–30, Edman N-terminal sequencing, cloning of a cDNA, and subsequent similar studies on full length PrPSc and

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PrPC. These methods revealed a 254-amino acid sequence from which an N-terminal 22-amino acid hydrophobic signal peptide is removed in the development of the mature proteins, which start at Lys23. The major start site for PrP 27–30 was known from Edman sequencing to be Gly90. The C-terminus was also known to be modified by the removal of another hydrophobic peptide of unknown length and the attachment of the GPI anchor. SDS-PAGE mobilities had shown that the majority of protein molecules carry oligosaccharides at both the consensus sites, Asn181 and Asn197. To confirm that the sequence of PrP was identical to that predicted from the gene sequence, it was necessary to cleave it selectively at specific residues to give a number of smaller peptide fragments. Hopefully these could be separated by HPLC and analyzed either off-line by collecting fractions or on-line by directly coupled HPLC-MS. The strategy adopted was to select an enzyme that would produce a relatively small number of peptides, the smaller of which would be analyzed directly and the larger would be digested with a second enzyme for further analysis. Originally it was intended to analyze all of the peptides by LSIMS, which in routine use has optimal sensitivity below 2500 Da but during the course of the work the further development of ESIMS allowed the direct analysis of much larger peptides. Endoproteinase Lys-C cleaving at the C-terminal side of lysine was selected as the first enzyme, for which PrP 27–30 should give nine fragments and full length PrP should give twelve fragments ranging from single lysines to a 75 amino acid glycopeptide His111-Lys185. In practice not all sites were cleaved with equal efficiency, (e.g., Lys-Pro bonds, of which there are three in PrP, are relatively stable and digestion was incomplete). Lys-C retains its activity in 0.1% sodium dodecyl sulfate (SDS) which is required to keep the protein in solution, even after denaturation and after removal of the GPI lipids with the enzyme PIPLC. SDS was subsequently removed by precipitation with guanidine as it interferes with most chromatographic and mass spectrometric methods. An HPLC chromatogram of a Lys-C digest of PrP 27–30 is illustrated in Fig. 1. LSIMS involves the ablation of cationized species such as protonated molecular ions from solution in a weakly acidic viscous liquid matrix of low volatility by bombardment with high energy cesium ions. This is very similar to FABMS, which uses argon or xenon atoms rather than cesium ions. A common matrix for both techniques is 1:1 glycerol/thioglycerol with 0.1% trifluoroacetic acid (TFA). In this study the ions were mass analyzed in a double-focusing sector mass spectrometer operating at ~3000 resolving power; thus peaks at adjacent mass numbers were clearly resolved, allowing mass measurement to ±0.2 Da. LSIMS was ideal for studying the midsize peptides of about 5 to 20

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FIG. 1. Reversed phase HPLC chromatogram of an endoproteinase Lys C digest of PrP 27–30, showing absorbance at 214 and 280 nm versus time (min). The gradient line shows acetonitrile content. The peptide compositions shown for each peak were derived from the experiments described in Section III. Peptides were identified containing all residues between 74 and 231, with the exception of an anticipated tetrapeptide, residues 107–110. (Reproduced with permission from Stahl, N. et al. in Prusiner, S.B., Collinge, J., Powell, J., and Anderton, B. [1992], Prion Diseases of Humans and Animals, pp. 361–379. Copyright Elsevier Science).

residues, seven of the nine predicted fragments from PrP 27–30 falling in this category. The 75 residue peptide was anticipated to be too large for this methodology and the tetrapeptide Thr-Asn-Met-Lys might be too small and hydrophilic for efficient detection. The primary information provided by mass spectrometry is molecular mass, measurements being made on each peak eluting from the HPLC. From the predicted protein sequence a computer program generated all possible cleavage products for any given enzyme, matching the measured and calculated masses. Any HPLC fraction giving a positive match could be further analyzed by amino acid analysis (AAA), MS/MS and/or Edman N-terminal sequencing. In this way the majority of the PrP peptides were identified and confirmed to have the sequences anticipated from the cDNA sequence (22). Some modifica-

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tions were observed. An early eluting fraction gave a strong peak at m/z 1016.3, in agreement with the calculated mass of the protonated peptide Gln186-Lys194. A slightly later fraction with the same composition by AAA gave a peak at m/z 999.3. The latter peptide gave no signal by Edman analysis, indicating a blocked N-terminus as was confirmed by MS/MS, which showed the first residue had been converted to pyroglutamic acid (23). The peptide corresponding to residues 195–204, Gly-Glu-Asn-PheThr-Glu-Thr-Asp-Ile-Lys was anticipated to be glycosylated at Asn197. A fraction isolated as a weak HPLC peak gave a signal at m/z 1153.5, corresponding to the peptide without oligosaccharide, which was confirmed by both MS/MS and Edman. A larger and slightly earlier eluting HPLC peak had the same amino acid analysis but gave no signal by LSIMS. Edman analysis confirmed the sequence except that there was no signal at the third cycle, presumably due to glycosylation of the asparagine. Treating the peptide with PNGase F removed the N-linked oligosaccharide and converted Asn197 to aspartic acid. This was observable by LSIMS as a 1 Da increase in the peptide m/z, in this instance to m/z 1154.5. MS/MS showed that the first two residues were unchanged, the so-called b2 ion for Gly-Glu being at m/z 187. The b3 ion moved from m/z 301 for Gly-Glu-Asn for the naturally unglycosylated peptide to m/z 302 for Gly-Glu-Asp of the PNGase F-treated glycopeptide, as shown in Fig. 2. A minor component (~15%) was identified at m/z 932.3 as a C-terminal peptide Glu221-Gly228 having no GPI anchor. This was not an anticipated Lys-C cleavage site and was attributed to a small fraction of PrP that was devoid of the GPI (24). A further HPLC fraction containing the major C-terminal peptide carrying the GPI group was tentatively identified by AAA, although this gave no signal by LSIMS. Potential chemical and enzymatic methods to simplify this complex structure are summarized in Fig. 3. The GPI was removed by overnight treatment with 50% HF at 4°C, which hydrolyzes any phosphodiester linkages or phosphate groups (25). LSIMS then gave the mass of the protonated peptide as 1354.5 Da, corresponding to Glu221-Ser231, the C-terminal serine carrying the residual ethanolamine remaining after hydrolysis of the GPI. Thus, consistent with earlier predictions (25), the previously unknown site of attachment of the GPI was identified as Ser231 (24). Experiments with synthetic peptides established that the hydrophilic tetrapeptide Thr-Asn-Met-Lys was not retained on the C18 HPLC column. The HPLC column flow-through from the Lys-C digest was derivatized to add a cholate group to the N-terminus. The increased hydrophobicity allowed the derivatized peptide to be repurified by HPLC and analyzed by

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FIG. 2. A portion of the tandem mass spectrum of the glycopeptide, residues 195–204, containing the second glycosylation site, after treatment with PNGase F. The mass difference of 115 Da between the b2 and b3 ions is attributable to aspartic acid as the third residue (codon 197) rather than asparagine. (Reproduced with permission from Baldwin, MA. et al. [1993]. Trends in Analytical Chemistry 12, 239–248. Copyright Elsevier Science.)

LSIMS. The positive charge of the cholate group also enhanced the LSIMS signal, which was observed at the predicted m/z 652.3 (22). In addition to experiencing difficulties with the C-terminal peptideGPI, there were two peptides that were too large to be directly amenable to analysis by LSIMS, the 75 residue peptide of PrP 27–30 and a large Nterminal portion of full-length PrPSc. However, about this time ESIMS and MALDIMS became available for the analysis of such peptides. ESIMS was carried out by injecting the analyte into a flowing liquid matrix of 1:1 acetonitrile/water containing 1% acetic acid, which was sprayed from a positively charged needle into a chamber at atmospheric pressure. The resulting charged droplets evaporated under the influence of a stream of nitrogen. Analyte molecules were multiply protonated by the acid medium and drawn through an aperture into the vacuum of the mass spectrometer. Because mass spectrometers separate according to mass/number of charges (m/z), it was possible to monitor highly charged ions with molecular weights greater than the nominal mass range of the mass spectrometer. Furthermore, isolated molecular ions were obtained in the gas phase without the requirement for the analyte to be volatile. Lys C digestion of the Lys-Pro bonds at Lys27, Lys101, and Lys104 was incomplete; and the N-terminal portion from full length PrPSc was man-

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FIG. 3. Chemical and enzymatic treatment to reduce the size and complexity of the GPI anchor for mass spectrometric analysis. PIPLC removed the acylalkylglycerol lipids, then endoproteinase Lys C cut the amino acid chain after Lys220, giving the C-terminal peptide attached to the phosphorylated glycan. Incubation with 50% aqueous HF was used to hydrolyze the phosphodiester bonds and to release the glycan and the peptide, which were separated by RP-HPLC and analyzed independently.

ifested as a series of closely related peptides poorly resolved by HPLC. Four peptides were successfully identified by ESIMS, with a mean difference between the predicted and measured masses of 1.2 Da or 0.015% (22). The largest was Arg25-Lys106 of molecular mass 8,294.8 Da, observed as a series of ions with between seven and thirteen charges in the range m/z 600 to 1200. Further digestion by trypsin cleaved at Arg37 and Arg47, allowing smaller peptides to be isolated and analyzed. Arg25 was found to be resistant to trypsin as it is followed by proline. Earlier Edman sequencing of the intact protein had suggested possible modification to Arg25 and Arg37, but this was not confirmed in the present study. It was subsequently suggested that the modification might be to citrulline (J. Hope, personal communication), the mass of which is only 1 Da higher than arginine. Because the purification of full-length PrPSc was less effective than that of PrP 27–30, the quality of the original data for the full length peptide made it difficult to distinguish unambiguously between the possibilities of arginine or citrulline at these two positions. However, trypsin would not digest at citrulline; therefore the observation of the expected shorter peptides in the trypsin digest con-

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firmed that at least a fraction of the molecules contained arginine rather than citrulline at residue 37. The tryptic peptide of residues 23–37, presumably containing Arg25, gave the expected mass for the presence of arginine rather than citrulline, although a substoichiometric amount of citrulline could be present, as the M+1 ion was more intense than would be expected based on stable isotope ratios. IV. NON-PrP PEPTIDES IN PRION PREPARATIONS In the purifcation of PrPSc from brain tissue, it was very difficult to eliminate all other proteins. Every PrPSc preparation gave peptide signals that could not be attributed to the PrP sequence. The contaminants were often at low level and frequently coincided in HPLC fractions with PrP peptides. The HPLC fraction containing the N-terminal peptide of PrP 27–30 Gly90-Lys101 (m/z 1283.5) from several different preparations was always accompanied by ions at m/z 772.3 and 915.4. Edman analysis confirmed the presence of a mixture of peptides but could not distinguish signals from the different components and gave only limited sequence data. By contrast MS/MS was used to study each individual component in the mixture, characterizing these particular peptides as Asp-Gly-Pro-Arg-Leu-Ser-Lys and Arg-Glu-Ile-Val-AspArg-Lys. The first of these showed some homology with the leucine zipper region of a Drosophila protein, and the second showed some homology to mouse actin but the hamster sequence had not been reported. It is unlikely that these or other unidentified peptides play any role in the development of scrapie. V. N-LINKED OLIGOSACCHARIDES Preliminary studies by Endo et al. identified some of the basic structures of the N-linked oligosaccharides (15). The ESIMS analysis of the peptide His111-Lys185 confirmed that it is modified by the addition of heterogeneous groups that increase the molecular mass by 1800 to 3000 Da. Figure 4A shows the ESIMS spectrum with a repeating cluster of ions carrying 7–10 charges. This was expected as this peptide includes the first glycosylation site at Asn181. This spectrum was transformed to convert the multiply charged ion profiles into molecular masses (Fig. 4B). After treatment with PNGase F a single component was obtained (not shown), the molecular mass of which was 8607.8 Da as measured by ESIMS, close to the predicted value of 8608.6 for the deglycosylated peptide with con-

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FIG. 4. ESIMS spectrum of the glycopeptide His111-Lys185. (A) Raw data showing clusters of multiply charged species with 7–10 protons. (B) Transformed spectrum showing molecular masses rather than m/z, with three predominant species of 10256, 10564, and 10873 Da. (Reproduced with permission from Baldwin, M.A. et al. [1993]. Trends in Analytical Chemistry 12, 239–248. Copyright Elsevier Science.)

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version of asparagine to aspartic acid (22). The differences between the masses before and after treatment with PNGase F, as measured both by ESIMS and MALDIMS, confirmed the identity of the major oligosaccharides that had been partly characterized in the previous study (15). Thus the most abundant glycosylated species of mass 10564.5 Da has an oligosaccharide of measured mass 1956.3 Da, within 0.3 Da of that calculated for the structure of composition Hex4.HexNAc5.Fuc2 previously identified as the major neutral sugar. A more recent comprehensive mass spectrometric study of the N-linked sugars of murine PrPSc involved separation of the two glycosylation sites by tryptic digestion (26). Glycopeptide mixtures from sequential exoglycosidase digestions with combinations of neuraminidase, alpha-mannosidase and β galactosidase, were characterized by HPLC-ESIMS and MS/MS. This identified approximately 60 different sugar structures on PrPSc and demonstrated the presence of larger and more complex sugars at Asn196 than at Asn180 (mouse numbering), as shown in Fig. 5. A separate study that compared the sugars of hamster PrPC and PrPSc involved a completely different methodology in which the sugars were released from the protein, N-acetylated, fluorescently labeled by reductive aminolysis, separated by chromatography and analyzed by MALDIMS (27). It was found that essentially the same array of sugars was present in both isoforms, even though the structures containing bisecting N-acetylglucosamine residues were diminished in abundance in hamster PrPSc relative to PrPC. This significant result implies that PrPSc is not derived from any special subset of specifically glycosylated PrPC, although the reduced levels of GlcNAc imply a decrease in the activity of N-acetyaminotransferase III in scrapieinfected cells. Coincidently, these two studies also showed the sugars of mouse and hamster PrPSc to be essentially the same. VI. ANALYSIS OF THE GPI ANCHOR PrP was first identified as a GPI-anchored protein by Stahl et al. (17). All GPI anchors that have been fully characterized conform to a common pattern with a sugar glycan core containing the sequence Man3.HexN.Inos phosphate, the nonreducing terminus of which is attached through phosphoethanolamine to the C-terminus of the protein. The inositol phosphate has a lipophilic tail containing acylalkylglycerol. There is variability in the attachment of further sugar units and mammalian anchors have a further phosphoethanolamine. The previously utilized analytical methodology for GPI anchors using radiolabeling by hydrazinolysis, exoglycosidase digestion and gel permeation

40

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chromatography was not sufficiently sensitive for the small scale analysis required for PrP; thus several new approaches were implemented during this study (28–30). A. ESIMS of the C-terminal Peptide-GPI The C-terminal peptide could not be analyzed by LSIMS whilst still attached to the GPI, whereas ESIMS of an aliquot of the unhydrolysed fraction revealed several molecular species (Fig. 6). Certain components of the GPI could be predicted by analogy with other known structures (i.e., the glycan core would be linked to the protein through phosphoethanolamine, there would probably be a second phosphoethanolamine group, and the core would contain at least three mannose residues linked to glucosamine and phosphoinositol). Acylalkylglycerol had been removed by the action of PIPLC. The mass spectrometric analysis of oligosaccharides differs from that of peptides and proteins for which the basic building blocks are the 20 naturally occurring amino acids, each having a unique mass. The basic units of oligosaccharides are mostly isomers of only four or five unique compositions; thus it is relatively easy to assign the composition of an oligosaccharide on the basis of the number of hexose units (mass 162), hexosamines (mass 161), Nacetylhexosamines (mass 203), sialic acids (mass 291), etc. The various molecular species revealed by ESIMS differing in mass by combinations of 162 and 291 Da were assigned carbohydrate compositions. The smallest was calculated to contain the peptide, two phosphoethanolamines, three hexoses, one hexosamine, one N-acetyl hexosamine and phosphoinositol (i.e., the basic mammalian GPI with an additional Nacetylhexosamine). Larger forms contained a further one or two hexose units and unexpectedly, sialic acid (N-acetylneuraminic acid), a sugar not previously reported as a component of a GPI (30). Thus mass spectrometry showed the GPI was heterogeneous with at least five separate species and its composition was consistent with known GPI structures but with sialic acid as a novel component. The heterogeneity was confirmed by various separative techniques including capillary electrophoresis, Dionex high performance anion exchange chromatography (HPAEC), and reverse phase HPLC at pH 7. Cleavage FIG. 5. Oligosaccharide structures for mouse PrPSc at Asn180 and 196 (equivalent to 181 and 197 in hamster and human), obtained by Stimson et al. (26), grouped as (I) biantennary, (II) triantennary, and (III) tetraantennary. Squares, N-acetylglucosamine; open circles, mannose; closed circles, galactose; triangles, fucose; ovals, sialic acid. (Reproduced with permission from Stimson, E. et al. [1999]. Biochemistry 38, 4885–4895. Copyright American Chemical Society.)

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FIG. 6. Transformed ESIMS spectrum of the heterogeneous GPI species attached to the C-terminal peptide Glu221-Ser231. A, (Peptide 221–231).Ea.P.Hex3.(HexNAc) (P.Ea).HexN.Ino.P, [Ea = ethanolamine; Hex, hexose; HexN, hexosamine; HexNAc, Nacetylhexosamine; I, inositol; P, phosphate; Sia, sialic acid.]

of sialic acid by neuraminidase was also demonstrated. ESIMS showed that only species with at least four hexose units were sensitive to Jack bean α mannosidase and then only one residue was removed, consistent with the third mannose away from the glucosamine being the site of attachment to the protein. Mass spectrometry was not able to identify the particular isomeric form of each sugar unit (e.g., hexoses can be glucose, mannose or galactose, differing in the configuration of the ring hydroxyl groups). However, the sugars were identified by acid hydrolysis of the GPI and separation of the various monosaccharides by HPAEC. Comparison with the monosaccharide standards confirmed 3 or 4 mannose units, the glucosamine and sialic acid. N-acetylgalactosamine and galactose were also identified (30). B. Branching Patterns in the GPI Glycan by MS/MS Oligosaccharides differ from peptides and proteins in that they are frequently branched. Branching patterns are usually determined by digestion with glycosidases with specific activities followed by chromatographic analysis of the products, comparing retention times with those of standards. However, standards may not be available for studies on novel structures, and this approach requires relatively large amounts of material. Nuclear magnetic resonance analysis is even less sensitive. By contrast mass spectrometry is extremely sensitive and it was anticipated that the fragmentation induced by CID might reveal the branching pat-

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terns. MS/MS also has the ability to study individual selected species in unseparated mixtures such as the heterogeneous GPI glycans. High-resolution MS/MS with high energy CID was carried out on the glycans released by 50% HF and permethylated to enhance surface activity and volatility and to give a positively charged quaternary ammonium cation as the glucosamine became triply methylated, greatly enhancing the sensitivity for mass spectrometric detection. After methylation the masses of the protonated molecular ions for the most abundant species were measured as 1312.5, 1557.6, and 1761.6 Da, corresponding to the permethylated glycans lacking sialic acid. The MS/MS spectra shown in Fig. 7 represent fragmentation of these three species, separated in the first mass analyzer of the tandem mass spectrometer and caused to undergo collisions with neutral gas atoms, the fragment ions being separated in the second mass analyzer. The key to the interpretation of the tandem spectra was a series of ring cleavages across the individual hexose residues (so-called X-ions) that terminated in an ion of m/z 510 for the glucosamine.inositol moiety. Ions representing the loss of successive sugars revealed which sugar was substituted (i.e., which was the branch site). Complementary information came from the oxonium ions (called B ions by analogy with the fragmentation of peptide ions) from charge-remote fragmentations giving single or multiple sugar units from the nonreducing end of each chain (28). This is shown in Fig. 8 for the largest GPI species having sialic acid, or N-acetylneuraminic acid (NANA). The oxonium ions at m/z 376.1, 580.2 and 825.3 confirmed the presence of a linear sequence of ions corresponding to NANA.Hex.HexNAc. Thus tandem mass spectrometry together with chromatographic and glycosidase data allowed the identification of the six species shown in Fig. 9 (30). The branching was clearly revealed by cleavages along each branch of a biantennary structure. One branch represented the normal GPI glycan core with a chain of either three or four hexose units attached to the GlcN.lnos. The branch point was at the hexose immediately adjacent to the glucosamine with a chain of one, two, or three sugars in the sequence Sia.Glc.GlcNAc. The calculated molecular masses listed in Figure 9, which include the mass of the C-terminal peptide, can be related to the experimentally determined values in the ESI spectrum in Fig. 7. C. Unanswered Questions Concerning the GPI A further difference between oligosaccharides and proteins arises from the presence of alternative sites for linking individual sugars together. The glycosidic bond from carbon-1 of the nonreducing residue in a disaccha-

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FIG. 7. Partial tandem mass spectra for the region above m/z 500 of permethylated GPI anchor glycans devoid of sialic acid, showing the fragmentation of m/z 1312, 1557 and 1761. (Reproduced with permission from Baldwin, M.A. et al. [1990]. Analytical Biochemistry 191, 174–182. Copyright Academic Press.)

ride can be linked to positions 2, 3, 4, or 6 of the reducing residue. Furthermore, the glycosidic bond at carbon-1 has two possible configurations giving either α or β anomers. Each of these differences may be crucial in terms of biochemical action, but mass spectrometry cannot be applied easily to their analysis. Permethylation and hydrolysis followed by peracetylation and reduction with sodium borodeuteride gives partially methylated alditol acetates specifically labeled at one end, which can be analyzed by GC-MS to reveal the linkage positions. Unfortunately this strategy is difficult to prosecute with less than 5 to 10 nmole of homogeneous starting material. There are glycosidases that are specific for both anomericity and linkage position that are valuable in dealing with a structure likely to conform to a previously identified pattern. In this way it was shown that the third mannose in the chain, the site of attachment of the GPI to the protein, becomes susceptible to hydrolysis by alpha-mannosi-

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FIG. 8. Tandem mass spectrum of a permethylated GPI glycan of m/z 2123, containing sialic acid (NANA). (Reproduced with permission from Baldwin, M.A. et al. in Prusiner, S.B., Collinge, J., Powell, J., and Anderton, B. [1992]. Prion Diseases of Humans and Animals, pp. 380–397. Copyright Elsevier Science).

dase only after HF treatment. Interestingly the middle mannose in the chain of three at the core of the GPI does not show the same sensitivity (30), even though previous studies on GPIs from other proteins showed α1–6 linked mannose at this point (31). VII. ANALYSIS OF INTACT PrP BY MALDIMS AND ESIMS Enzyme digestion and peptide mapping are powerful methods for identifying the sites and the nature of any posttranslational modifications. However, measuring the intact molecular mass of the protein is the most effective way of ensuring that nothing was overlooked during the mapping experiments. ESIMS and MALDIMS are both capable of ionizing and accurately determining the mass of intact proteins. ESIMS generally offers higher resolution but for heterogeneous samples such as multiply glycosylated proteins, the presence of overlapping series of highly charged ions complicates the spectral analysis. MALDIMS is unable to resolve all the individual molecular species for a protein such as PrP, but it can provide the centroid or peak top mass of the unresolved peak profile. Samples for MALDIMS are dissolved in a solution containing a 104 to 105 molar excess of a UV-absorbing matrix, normally an aromatic acid

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FIG. 9. Structures of the various GPI anchors derived from experiments described in Section VI. The calculated masses include the C-terminal peptide, and can be directly correlated with the masses shown in Figure 5. Man, mannose; Gal, galactose; GalNAc, Nacetyl galactosamine; GlcNH2, glucosamine; Ino, inositol; Sia, sialic acid. (Reproduced with permission from Stahl, N. et al. [1999]. Biochemistry 31, 5043–5053. Copyright American Chemical Society.)

with additional conjugated double bonds such as 4-hydroxy-α-cyanocinnamic acid (4-HCCA). Without detergents or denaturants that suppress the ionization process, membrane proteins such as PrP are generally insoluble in solvents that are suitable for the matrix compound. PrPSc

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was treated with PNGase F to remove the N-linked sugars and reduce the heterogeneity, then dissolved in hexafluoroisopropanol and mixed 1:1 with a solution of 5 mg/mL 4-HCCA in 2:5:2 chloroform/ methanol/0.1% TFA. One µL of this mixture containing approximately 1 pmol of protein was deposited on the MALDI target and the solvent was evaporated. In the B. T. Chait’s laboratory at Rockefeller University, this was irradiated and ionized with 354 nm radiation from a Nd-YAG laser, the ablated ions being mass analyzed by time of flight mass spectrometry. The mass spectrum showed broad peaks corresponding to singly, doubly, and triply charged ions, and gave an average mass of ~25,350 Da (32). Taking account of the relative abundance of the different GPIs, the molecular masses for amino acids Lys23-Ser231 combined with the various GPI species (Fig. 9) and the likely GPI lipids, a weighted mean of 25,329 Da was calculated. Thus there was relatively good agreement between the calculated and measured numbers, suggesting that no major modification had been destroyed or overlooked. However, the resolving power of MALDIMS was not sufficient to separate the various forms present owing to the heterogeneity of the GPI anchor, hence the difficulty of measuring the molecular weight with high precision. ESIMS gives higher resolution, but it relies on the protein being soluble; PrPC is more soluble than PrPSc, and Fig. 9 compares more recent spectra of PrPC obtained by MALDIMS and ESIMS. Despite the removal of the sugars with PNGase F to reduce the overall heterogeneity, the heterogeneous GPI glycan remained attached. The protein was also treated with PIPLC to remove the lipids, thereby enhancing solubility in the ESIMS buffer of 1:1 acetonitrile water with 1% acetic acid. The upper spectrum shows the MALDIMS data obtained with a commercial mass spectrometer at UCSF for hamster brain PrPC mixed with myoglobin as an internal mass calibrant. The PrP peaks are broader than those of myoglobin and show unresolved structure. Using peak tops to define the mass of each ion, PrPC appeared as a singly charged ion at 24,466.1, doubly charged at 12,236.9 and triply charged at 8,161.6. The mean molecular mass calculated from these three species was 24,472.9 Da. Note that it is generally more accurate to use centroids rather than peak tops, but not in the case of unresolved peaks arising from ions of different compositions. Because of much higher charge states, a spectrum of the same sample from ESIMS in the lower panel appears to be completely different. Peaks are observed for the attachment of between 20 and 32 protons, each charge state showing at least two major species. The raw data on the left was deconvoluted to the molecular weight pattern shown in the right hand panel. Here the different glycoforms of the GPI are well resolved and give a molecular weight for the major species of 24,474.0. The calcu-

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lated value for the PrP sequence plus the major GPI glycoform is 24,474.5 Da, in excellent agreement with the ESIMS result (0.002%) and in surprisingly good agreement with MALDIMS, considering the poorly resolved nature of the MALDI spectrum. VIII. PROCESSING OF CHICKEN PrP Chicken PrP shows moderate homolgy to mammalian prion proteins. Harris et al. (33) used sequence-specific antibodies to immunoprecipitate and immunoblot chicken PrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. They used MALDIMS to characterize the protein fragments indicative of natural processing sites. The majority of chicken PrP protein molecules present in neuroblastoma cells and on isolated brain membranes are attached to the cell surface by the GPI anchor. Most of these surface-anchored molecules were found to be truncated at their N-termini, distal to the proline/glycine-rich repeats. Corresponding N-terminal fragments found in the medium were identified by mass spectrometry, such as a peptide extending from Lys25 to Phe116. Such cleavages were localized within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that could have physiological as well as pathological significance. An alternative cleavage site within the GPI anchor caused the release of a fraction of the membrane-bound protein into the medium. IX. RECOMBINANT PrP AND SYNTHETIC PEPTIDES There have been numerous reports of the use of mass spectrometry to analyze recombinant PrP and synthetic peptides corresponding to regions of the PrP sequence. Most of these reports have not been reviewed here, as the methodolgy is well established and in general has been used purely to confirm the identity of the species.

FIG. 10. Mass spectra of PrPSc from MALDIMS, and from ESIMS. For MALDIMS the mass scale was calibrated with myoglobin ions as an internal standard. For ESIMS the mass scale was calibrated externally using the doubly charged and singly charged ions of gramicidin S. The right hand panel of the ESIMS spectrum shows the deconvoluted spectrum, giving measured molecular weights for the major species, which differ by one hexose. Deviations from the calculated values are shown in parentheses.

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A. PrP Modifications Caused by Aging Prion diseases are associated with aging, and the incidence of certain protein modifications is known to increase with age. One such modification is the conversion of asparagine to aspartic acid and isoaspartic acid. It is possible that the conversion of PrPC to PrPSc might be enhanced by the presence of such modified amino acids. After aging recombinant mouse PrP at 37°C, 0.8 mole of isoaspartyl residue per mole of protein was detected by a protein-l-isoaspartyl methyltransferase assay, with a half-life for conversion of 30 days–1. Digestion of the modified PrP with endoproteinase Lys C, followed by mass spectrometry and Edman degradation, identified Asn108 in the amino-terminal flexible region to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226 (34). However, despite an independent search for such modifications in PrPSc, they have yet to be identified in significant amounts (35), although it remains possible that such modifications in substoichiometric numbers of PrP molecules could help to initiate the PrPSc formation or stabilize PrPSc polymers in vivo. B. Copper Binding PrP-knockout mice apparently develop normally and are noteworthy only for their resistance to infection by prions; thus the normal function of PrPC is unclear. Nevertheless, there is an increasing body of evidence that it is involved in either transport or storage of copper. One of the first pieces of evidence was the demonstration by MALDIMS and ESIMS that synthetic peptides containing multiple copies of the PrP octarepeat ProHisGlyGlyGlyTrpGlyGln selectively bind copper(II) ions, but not other divalent cations (36, 37). Mass spectrometry, particularly ESIMS, is increasingly accepted as a valid means for studying noncovalent associations, including those between proteins and metal ions (38, 39). ESIMS has been used to demonstrate that the stoichiometries of the complexes are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity, particularly for peptides extended beyond the octarepeats to incorporate His96. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides were shown to be in the nanomolar to micromolar range (40). PrPC is known to concentrate in cell surface lipid-rich caveolae, which can be endocytosed as endosomes and secondary lysosomes at reduced pH. Thus PrP could function as a Cu2+ transporter, binding Cu2+ ions from the extracellular medium under

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physiological conditions and then releasing some or all of this metal within the cell on exposure to acidic pH. X. ACCESSORY MOLECULES IN SCRAPIE PRIONS Molecular biological and transgenic mouse experiments have demonstrated virtually beyond doubt that an isoform of PrP is the pathogenic agent in the TSEs. However, there have long been questions as to whether an accessory molecule is another essential component of infectious prions. One justification for this question is the failure to either regenerate infectivity in denatured PrP or create infectivity in synthetic or recombinant material. A number of candidate molecule classes have been considered, including other proteins, nucleic acids, glycosaminoglycans, sugars, and lipids. In general, the effectiveness of MALDIMS analysis of lipids is less predictable than for the analysis of peptides and proteins. Nevertheless, a combination of thin layer chromatography and MALDIMS was used to demonstrate that prion rods contain two host sphingolipids (41). Samples dried from chloroform with 5% methanol onto the MALDI target that had been pretreated with the matrix compound 2,5-dihydroxybenzoic acid gave normal mass spectra and fragment spectra using a technique known as post-source decay (PSD). In this way, it was established that chloroform/methanol extraction routinely yielded galactosylceramide and sphingomyelin, although in lower molar abundance than PrP. It is highly probable that these lipids were extracted from brain during the purification of PrPSc, which is known to be associated with membrane fractions, but it is not known whether they play any role in prion infectivity. The same group has recently identified the presence of an insoluble and inert polysaccharide scaffold in prion rods, remaining after prolonged protease digestion had removed at least 99.8% of PrP (42). The identification and analysis, which used direct chemical ionization, hydrolysis, conversion of the polysaccharide to alditol acetates and GCMS, showed predominantly 1,4-linked glucose structures. Such findings are relatively ubiquitous by this methodology; thus it is uncertain whether the presence of this polysaccharide in prions has any structural or functional significance. XI. CONCLUSIONS Mass spectrometry offers an array of powerful techniques for the analysis of the primary structure of proteins. By using a combination of

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these methods, PrPSc was confirmed to have an amino acid sequence identical to that predicted from the gene sequence. The nature of the posttranslational modifications was also delineated. Preliminary data on the heterogeneous N-linked sugars was substantiated by recent comprehensive studies that suggest that there are no unique components responsible for PrPSc formation. The GPI anchor was subjected to extensive analysis, revealing certain features never previously reported for comparable structures. However, further experiments suggested that these features are also present in PrPC and therefore do not provide the basis for an explanation of the different properties of PrPSc. Non-PrP peptides and other accessory molecules have been identified, but it appears unlikely that these are of any significance. The most difficult question that remains unanswered is whether a small fraction of molecules carrying a crucial modification has been overlooked. This is possible as one infectious prion unit is known to contain tens of thousands of PrP molecules. However, in summary, the weight of evidence from mass spectrometry supports the notion that the difference between PrPSc and PrPC underlying the infectivity of prions is not chemical but is essentially conformational (43). ACKNOWLEDGMENTS Mass spectrometry carried out in the UCSF Mass Spectrometry Facility was supported by NIH RR01614.

REFERENCES 1. Barber, M., Bordoli, R.S., Sedgewick, R.D., and Tyler, A.N.J. (1981). J. Chem. Soc., Chem. Commun. 325–327. 2. Aberth, W., Straub, K., and Burlingame, A.L. (1982). Anal. Chem. 54, 2029–2034. 3. McCloskey, J.A. ed., (1991). Meth. Enzymol. Vol. 193, Academic Press, San Diego. 4. Wong, S.F., Meng, C.K., and Fenn, J.B. (1988). J. Phys. Chem. 92, 546–550. 5. Fenn, J.B., Mann, M., Meng, C.K., and Wong, S.K. (1990). Mass Spectrom. Rev. 9, 37–70. 6. Karas, M., and Hillenkamp, F. (1988). Anal. Chem. 60, 2299–2301. 7. Karas, M., Bahr, U., and Hillenkamp, F. (1989). Int. J. Mass Spectrom. Ion Processes 92, 231–242. 8. Karas, M., Bahr, U., Ingedoh, A., and Hillenkamp, F. (1989). Angew. Chem. Intl. Ed. Engl. 28, 760–761. 9. Chait, B.T., and Kent, S.B. (1992). Science 257, 1885–1894. 10. Burlingame, A.L., Carr, S.A., and Baldwin, M.A. Eds. (2000). “Mass Spectrometry in Biology and Medicine”, Totowa, NJ: Humana. 11. Oesch, B., Westaway, D., Wälchli, M., McKinley, M.P., Kent, S.B.H., Aebersold, R., Barry, R.A., Tempst, P., Teplow, D.B., Hood, L.E., Prusiner, S.B., and Weissmann, C. (1985). Cell 40, 735–746.

MASS SPECTROMETRIC ANALYSIS OF PRION PROTEINS

53

12. Basler, K., Oesch, B., Scott, M., Westaway, D., Wälchli, M., Groth, D.F., McKinley, M.P., Prusiner, S.B., and Weissmann, C. (1986). Cell 46, 417–428. 13. Hope, J., Morton, L.J.D., Farquhar, C.F., Multhaup, G., Beyreuther, K., and Kimberlin, R. (1986). EMBO J. 5, 2591–2597. 14. Turk, E., Teplow, D.B., Hood, L.E., and Prusiner, S.B. (1988). Eur. J. Biochem. 176, 21–30. 15. Endo, T., Groth, D., Prusiner, S.B., and Kobata, A. (1989). Biochemistry 28, 8380–8388. 16. Haraguchi, T., Fisher, S., Olofsson, S., Endo, T., Groth, D., Tarentino, A., Borchelt, D.R., Teplow, D., Hood, L., Burlingame, A.L., Lycke, E., Kobata, A., and Prusiner, S.B. (1989). Arch. Biochem. Biophys. 274, 1–13. 17. Stahl, N., Borchelt, D.R., Hsiao, K., and Prusiner, S.B. (1987). Cell 51, 229–240. 18. Bolton, D.C., McKinley, M.P., and Prusiner, S.B. (1982). Science 218, 1309–1311. 19. Prusiner, S.B., Bolton, D.C., Groth, D.F., Bowman, K.A., Cochran, S.P., and McKinley, M.P. (1982). Biochemistry 21, 6942–6950. 20. McKinley, M.P., Bolton, D.C., and Prusiner, S.B. (1983). Cell 35, 57–62. 21. Pan, K.-M., Stahl, N., and Prusiner, S.B. (1992). Protein Sci. 1, 1343–1352. 22. Stahl, N., Baldwin, M.A., Teplow, D., Hood, L., Gibson, B.W., Burlingame, A.L., and Prusiner, S.B. (1993). Biochemistry 32, 1991–2002. 23. Baldwin, M.A., Falick, A.M., Gibson, B.W., Prusiner, S.B., Stahl, N., and Burlingame, A.L. (1990). J. Am. Soc. Mass Spectrom 1, 258–264. 24. Stahl, N., Baldwin, M.A., Burlingame, A.L., and Prusiner, S.B. (1990). Biochemistry 29, 8879–8884. 25. Ferguson, M.A.J., and Williams, A.F. (1988). Annu. Rev. Biochem. 57, 285–320. 26. Stimson, E., Hope, J., Chong, A. and Burlingame, A.L. (1999). Biochemistry 38, 4885–4895. 27. Rudd, P.M., Endo, T., Colominas, C., Groth, D., Wheeler, S.F., Harvey, D.J., Wormald, M.R., Serban, H., Prusiner, S.B., Kobata A., and Dwek, R.A. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 13044–13049. 28. Baldwin, M.A., Stahl, N., Reinders, L.G., Gibson, B.W., Prusiner, S.B., and Burlingame, A.L. (1990). Anal. Biochem. 191, 174–182. 29. Baldwin, M.A., Stahl, N., Burlingame, A.L., and Prusiner, S.B. (1990). In “Methods: A Companion to Methods in Enzymology” 1, 306–314. Academic Press, San Diego. 30. Stahl, N., Baldwin, M.A., Hecker, R., Pan, K.-M., Burlingame, A.L., and Prusiner, S.B. (1992). Biochemistry 31, 5043–5053. 31. Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988). Science 239, 753–759. 32. Baldwin, M.A., Wang, R., Pan, K.-M., Hecker, R., Stahl, N., Chait, B.T, and Prusiner, S.B. (1993). In “Techniques in Protein Chemistry” (R. Hogue Angeletti, ed.) Vol. IV, Academic Press, San Diego. pp. 41–45. 33. Harris, D.A., Huber, M.T., van Dijken, P., Shyng, S.L., Chait, B.T., and Wang, R. (1993). Biochemistry 32, 1009–1016. 34. Sandmeier, E., Hunziker, P., Kunz, B., Sack, R., and Christen. P. (1999). Biochem. Biophys. Res. Commun. 32, 1009–1016. 35. Weber, D.J., McFadden, P.N., and Caughey, B. (1998). Biochem. Biophys. Res. Commun. 246, 606–608. 36. Hornshaw, M.P., McDermott, J.R., and Candy, J.M. (1995). Biochem. Biophys. Res. Commun. 207, 621–629. 37. Hornshaw, M.P., McDermott, J.R., Candy, J.M., and Lakey, J.H. (1995). Biochem. Biophys. Res. Commun. 214, 993–999. 38. Loo, J.A. (1997). Mass Spectrom. Rev. 16, 1–23. 39. Last, A.M., and Robinson, C.V. (1999). Curr. Opin. Chem. Biol. 3, 564–570.

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40. Whittal, R.M., Ball, H.L., Cohen, F.E., Burlingame, A.L., Prusiner, S.B., and Baldwin, M.A. (2000). Protein Sci. 9, 332–343. 41. Klein, T.R., Kiersch, D., Kaufmann, R., and Riesner, D. (1998). Biol. Chem. 379, 655–666. 42. Appel, T.R., Dumpitak, C., Matthiesen, U., and Riesner, D. (1999). Biol. Chem. 380, 1295–1306. 43. Cohen, F.E., Pan, K.-M., Huang, Z., Baldwin, M.A., Fletterick, R.J., and Prusiner, S.B. (1994). Science 264, 530–531.