Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.
SummaryThe HPr kinase of Gram-positive bacteria is an ATPdependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-SerHPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/ phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.
The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.
In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.
Following hints from X-ray data~Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547-10553;Yoshikawa S et al., 1998, Science 280:1723-1729!, chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copper B -coordinated His240-Tyr244! cross-link at the O 2 -activating Heme Fea 3 -Cu B center in the catalytic subunit I of the enzyme. The early evolutionary invention of this unusual structure may have prevented demaging• OH-radical release at e Ϫ -transfer to dioxygen and thus have enabled O 2 respiration.
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