Time of Detection of Prions in the Brain by Nanoscale Liquid Chromatography Coupled to Tandem Mass Spectrometry Is Comparable to Animal Bioassay.

Prions cause transmissible and inevitably fatal neurological diseases in agriculturally important animals, including bovine spongiform encephalopathy in domestic cattle, scrapie in sheep and goats, and chronic wasting disease in cervids. Because animals are largely asymptomatic throughout the course of the disease, early detection of prion disease is important. Hamsters were peripherally (ip) inoculated with hamster-adapted (Sc237) prions. By week 13 of a 14-week disease course, clinical signs appeared. A multiple-reaction-monitoring-based method was used to quantitate the amount of proteinase-K-digested prions (PrP 27-30) and the extent of methionine 213 oxidation present in the brains of infected hamsters. Detectable amounts of PrP 27-30 were present in all animals after 4 weeks. The extent of methionine 213 oxidation decreased over time. When we compared our quantitation results to those from other researchers using bioassay, we observed that consistent detection of PrP 27-30 by mass spectrometry occurs at a time when prions are reliably detected by bioassay.

Quantifying the Role of Lysine in Prion Replication by Nano-LC Mass Spectrometry and Bioassay.

Prions propagate by a template driven process, inducing the normal cellular isoform (PrPC) to adopt the prion (PrPSc) conformation. In PrPC, the positions of lysines are highly conserved and strongly influence prion propagation. In this study, covalent modification was used to quantitate the role of lysines in the PrPSc template that drives prion replication. The ε-amino group of lysines in the PrPSc (hamster-adapted scrapie Sc237) template was acetylated by either acetic anhydride (Ac2O) or the N-hydroxysuccinimide ester of acetic acid (Ac-NHS). The extent of lysine acetylation in PrPSc was quantitated by mass spectrometry or Western blot-based analysis. Identical samples were bioassayed to quantitate the loss of infectivity associated with lysine acetylation. The reduction of infectivity at the highest reagent concentration was approximately 90% (∼10-fold). Ten of the eleven prion lysines were acetylated to a greater extent (25-400-fold) than the observed loss of infectivity. Only one lysine, at position 220 (K220), had a reactivity that is consistent with the loss of infectivity. Although lysines are highly conserved and play a crucial role in converting PrPC into the PrPSc conformation, once that conformation is adopted, the lysines present in the PrPSc template play only a limited role in prion replication. In principle, this approach could be used to clarify the role of other amino acids in the replication of prions and other prion-like protein misfolding diseases.

Covalent Surface Modification of Prions: A Mass Spectrometry-Based Means of Detecting Distinctive Structural Features of Prion Strains.

Prions (PrP(Sc)) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrP(C)) into a prion. The only demonstrated difference between PrP(C) and PrP(Sc) is conformational: they are isoforms. A given host can be infected by more than one kind or strain of prion. Five strains of hamster-adapted scrapie [Sc237 (=263K), drowsy, 139H, 22AH, and 22CH] and recombinant PrP were reacted with five different concentrations (0, 1, 5, 10, and 20 mM) of reagent (N-hydroxysuccinimide ester of acetic acid) that acetylates lysines. The extent of lysine acetylation was quantitated by mass spectrometry. The lysines in rPrP react similarly. The lysines in the strains react differently from one another in a given strain and react differently when strains are compared. Lysines in the C-terminal region of prions have different strain-dependent reactivity. The results are consistent with a recently proposed model for the structure of a prion. This model proposes that prions are composed of a four-rung ß-solenoid structure comprised of four ß-sheets that are joined by loops and turns of amino acids. Variation in the amino acid composition of the loops and ß-sheet structures is thought to result in different strains of prions.

Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 µg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.

Structural organization of mammalian prions as probed by limited proteolysis.

Elucidation of the structure of PrP(Sc) continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP(Sc)). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP(Sc) from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP(Sc) samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP(Sc). These results reinforce the hypothesis that the structure of PrP(Sc) consists of a series of highly PK-resistant ß-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP(Sc) is highly resistant to PK and therefore perhaps also contains ß-sheet secondary structure.

Human and rat brain lipofuscin proteome.

The accumulation of an autofluorescent pigment called lipofuscin in neurons is an invariable hallmark of brain aging. So far, this material has been considered to be waste material without particular relevance for cellular pathology. However, two lines of evidence argue that lipofuscin may play a yet unidentified role for pathological cellular functions: (i) Genetic forms of premature accumulation of similar autofluorescent material in neuronal ceroid lipofuscinosis indicate a direct disease-associated link to lipofuscin; (ii) Retinal pigment epithelium cell lipofuscin is mechanistically linked to age-associated macular degeneration. Here, we purified autofluorescent material from the temporal and hippocampal cortices of three different human individuals by a two-step ultracentrifugation on sucrose gradients. For human brain lipofuscin, we could identify a common set of 49 (among > 200 total) proteins that are mainly derived from mitochondria, cytoskeleton, and cell membrane. This brain lipofuscin proteome was validated in an interspecies comparison with whole brain rat lipofuscin (total > 300 proteins), purified by the same procedure, yielding an overlap of 32 proteins (64%) between lipofuscins of both species. Our study is the first to characterize human and rat brain lipofuscin and identifies high homology, pointing to common cellular pathomechanisms of age-associated lipofuscin accumulation despite the huge (40-fold) difference in the lifespan of these species. Our identification of these distinct proteins will now allow research in disturbed molecular pathways during age-associated dysfunctional lysosomal degradation.

PK-sensitive PrP is infectious and shares basic structural features with PK-resistant PrP.

One of the main characteristics of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP(Sc) that is sensitive to PK hydrolysis (sPrP(Sc)). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP(Sc) versus the PK-resistant (rPrP(Sc)) fractions of PrP(Sc) and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP(Sc) and rPrP(Sc) fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP(Sc) to the rPrP(Sc) forms of PrP(Sc). Although the sPrP(Sc) and rPrP(Sc) fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.

Utility of mass spectrometry in the diagnosis of prion diseases.

We developed a sensitive mass spectrometry-based 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.

Assessing the role of oxidized methionine at position 213 in the formation of prions in hamsters.

Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-translational modifications and differ solely in conformation. Recent work has suggested that the oxidized form of the methionine at position 213 (Met213) plays a role in the conversion of the normal cellular prion protein to the prion conformation and is a prion-specific covalent signature. We developed a sensitive method of quantitating the methionine sulfoxide present at position 213 (MetSO213) and used this method to measure the changes in MetSO213 over the time course of an intracranial challenge, using the 263K strain of hamster-adapted scrapie. These results indicate that the proportion of Met213 that is oxidized decreases over the course of the disease. We examined the quantity of MetSO213 in PrP(C) and compared it to the amount found in animals terminally afflicted with the 263K, 139H, and drowsy strains of hamster-adapted scrapie. These strains show only low levels of MetSO213 that is comparable to that of PrP(C). These data suggest that MetSO213 does not appear to be a prion-specific covalent signature.

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry.

Elucidation of the structure of scrapie prion protein (PrP(Sc)), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP(Sc). Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in beta-strands. We treated PrP(Sc) isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP(Sc) was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP(Sc) confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP(Sc) are susceptible to PK. Our results indicate that besides the "classic" amino-terminal PK cleavage points, PrP(Sc) contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short beta-sheet stretches connected by loops and turns.

Sensitive, preclinical detection of prions in brain by nanospray liquid chromatography/tandem mass spectrometry.

More sensitive detection of prions in brain is important because it would allow early detection of disease in young animals and assure a safer food supply. We have quantitated the amount of proteinase K-resistant prion protein (PrP 27-30) by use of nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode of operation. We used a method based on the detection of VVEQMCTTQYQK (residues 209-220) obtained by reduction, alkylation and digestion with trypsin. Quantitation of the amount of PrP 27-30 in the brains of Syrian hamsters was possible as early as 24 h after inoculation. Our results show sensitive detection of 180 fmol of PrP 27-30 per g brain (wet weight) as early as 24 h after inoculation. Clinical symptoms are not observed until 9 weeks after inoculation.

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS.

Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein. Detection of VVEQMCTTQYQK was more sensitive than for the derivative with phenylisothiocyanate (PITC) because of decreased ionization efficiency of the PITC-derivatized peptides. The VVEQMCTTQYQK method has a LOD of 20 to 30 amol for pure standards. Proof of principle is demonstrated by quantitation of the amount of PrP 27-30 in the brains of terminally ill Syrian hamsters.