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.

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.

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.

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.

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.