Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation.

Epidermal growth factor-like domains (EGFDs) have important functions in cell-cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-ß-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry-based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.

The Hydroxyproline Proteome of HeLa Cells with Emphasis on the Active Sites of Protein Disulfide Isomerases.

Hydroxyproline-containing proteins (other than collagens) are rare and difficult to identify. Only 14 such proteins have been found in the human proteome by biochemical methods despite the fact that the list includes examples of biological importance such as hypoxia-inducible factor and the 40S ribosomal protein S23 (RPS23), both of which have significant biological function of the post-translational modification. Comparison of multinotch search software Global-PTM-Discovery to conventional proteomic database search software gave a nine-fold improvement in correctly identifying non-collagen peptides containing hydroxyproline. Manual interpretation of MS-MS spectra refined this list to discover 36 unique peptides representing 24 unique hydroxyproline sites in 21 proteins, of which only Pro62 of RPS23 had been reported previously in UniProt. Eight of the sites were found to be conserved as prolines in nine species examined, ranging from humans to yeast. These include sites 51 and 395 in protein disulfide-isomerase (PDIA1) and sites 204 and 553 in protein disulfide-isomerase A4 (PDIA4). The apparent occupancy of these sites ranged from 72-89%, suggesting a structural and possibly functional role of these PTMs. Fifteen of the sites most likely contain 4R-hydroxyproline (Pro30 of serpin H1, Pro520 of aspartyl/asparaginyl ß-hydroxylase, Pro223 of neutral α-glucosidase AB, Pro977 of hypoxia upregulated protein 1, Pro378 of protein ERGIC-53, Pro252 of protein CASC4, Pro545 of bromodomain-containing protein 2, Pro488 of bromodomain-containing protein 3, Pro130 of nucleolar RNA helicase 2, Pro51 of PDIA1, Pro395 of PDIA1, Pro404 of PDIA3, Pro89 of PDIA4, Pro204 of PDIA4, and Pro553 of PDIA4). The remaining sites could be either 4R-hydroxyproline or 3S-hydroxyproline. Recommendations are made to improve automated interpretation of proteomic data to improve future proteomic research whose goal is to mine more of the remaining dark matter of the proteome.

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.

A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

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.

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.

A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex.

We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence Summary: A host-targeted drug to treat all respiratory viruses without viral resistance development.

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.

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.

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.

Effects of purification on the bioavailability of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.

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.

Isolation and characterization of a proteinase K-sensitive PrPSc fraction.

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.

Proteinase K and the structure of PrPSc: The good, the bad and the ugly.

Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic 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) by Western blot, ELISA or immunohistochemical detection. PK digestion has also been used to detect differences in prion strains. Thus, PK has been a crucial tool to detect and, thereby, control the spread of prions. PK has also been used as a tool to probe the structure of PrP(Sc). Mass spectrometry and antibodies have been used to identify PK cleavage sites in PrP(Sc). These results have been used to identify the more accessible, flexible stretches connecting the ß-strand components in PrP(Sc). These data, combined with physical constraints imposed by spectroscopic results, were used to propose a qualitative model for the structure of PrP(Sc). Assuming that PrP(Sc) is a four rung ß-solenoid, we have threaded the PrP sequence to satisfy the PK proteolysis data and other experimental constraints.

Radiochemical detection for packed capillary liquid chromatography-mass spectrometry.

For the identification of trace level organic molecules, such as drug or pesticide metabolites, there is need of a practical method to do packed capillary liquid chromatography-mass spectrometry (LC-MS) with radiochemical detection. This problem has been successfully solved by use of a post column flow-splitter, with coaxially transported makeup flow that increases the split flow rate to a flow compatible with commercially available radiochemical flow cells. To test the device, 14C-labeled azoxystrobin, a commercial fungicide, was analyzed by liquid chromatography-radiochemical activity monitor-mass spectrometry (LC-RAM-MS) using a 0.32 mm i.d. packed capillary column. Azoxystrobin could be detected at 500 pCi with good signal/noise. The method is general and can be used with capillary LC columns of smaller diameters. Column efficiency of about 20,000 theoretical plates/m was achieved using either radiochemical or mass spectrometric data, thus demonstrating the lack of band broadening using the described method for radiochemical detection. The simple hardware described allows the routine use of packed capillary LC with radiochemical detection.

Identification of fonofos metabolites in Latuca sativa, Beta vulgaris, and Triticum aestivum by packed capillary flow fast atom bombardment tandem mass spectrometry.

The metabolism of fonofos, a thiophosphonate insecticide, was investigated in mature lettuce (Latuca sativa), beet (Beta vulgaris), and wheat (Triticum aestivum). Six new metabolites were identified by LC-MS and LC-MS-MS analysis using fast atom bombardment (FAB) and packed capillary LC columns with application of the on-column focusing technique. These methods provided the sensitivity required to identify unknown metabolites that were present in the mature plants at only 20-230 ppb. Structural elucidation was facilitated by use of fonofos labeled with both carbon-14 and carbon-13 in the phenyl ring. In all three plants fonofos was converted to a glucose conjugate of thiophenoxylactic acid. Oxidation of the glucose conjugate gave isomeric sulfoxides in all species examined. Thiophenoxylactic acid was found esterified to malonic acid in lettuce. In beets, S-phenylcysteine was found as its malonic acid amide. A second metabolite unique to beets was N-(malonyl)-[2[(ethoxyethylphosphinothionyl)oxy]phenyl]cysteine. This novel structure was confirmed by synthesis.

Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression.

The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of ß-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.

The nuclear transcription factor RARalpha associates with neuronal RNA granules and suppresses translation.

All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II alpha and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunit GluR1. Hippocampal neurons express RARalpha in dendrites, and knocking down RARalpha prevents all-trans-retinoic acid effects on dendritic growth. Here we show, by liquid chromatography/mass spectrometry analysis of immunoaffinity isolates of hippocampal neurons, that RARalpha partners with many RNA-binding proteins and translation factors conveyed in dendritic RNA transport granules, including the purine-rich element-binding protein, Pur alpha. The interaction of RARalpha with Pur alpha, an RNA-binding protein required for dendritic RNA transport, and other RNA-binding proteins was confirmed by tandem affinity purification. Confocal microscopy confirmed localization of neuronal RARalpha in dendritic RNA granules with Pur alpha and FMRP (the fragile x mental retardation protein). Hippocampal RARalpha also associates with mRNA, e.g. encoding GluR1 and calcium calmodulin-dependent protein kinase II alpha. Consistent with a granule function of conveying translationally silenced mRNA, RARalpha inhibits translation initiation, independent of 7-methylguanylate cap or poly(A) tail, and prompts mRNA redistribution to silencing ribonucleoprotein particles. These data afford a mechanism for rapid stimulation of dendritic growth by all-trans-retinoic acid and reveal that the ligand-dependent transcription factor RARalpha also regulates translation.

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.