The Stk1/Stp1 and GraSR signal-transduction pathways are two distinct pathways in Staphylococcus aureus that rely on a reversible phosphorylation process in transducing external stimuli intracellularly. Stk1/Stp1 is an eukaryote-like Ser/Thr kinase phosphatase pair involved in purine biosynthesis, cell-wall metabolism, and autolysis. GraSR is a two-component system involved in resistance to cationic antimicrobial peptides. Both systems are implicated in S. aureus virulence and resistance to cell-wall inhibitors. Our study shows that the response regulator protein GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA-binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR as well as the amino acid sequences flanking the phosphorylation sites. Its homologue in Bacillus subtilis , BceR, which harbors two of the three phosphorylation sites in GraR, does not undergo Stk1-dependent phosphorylation. GraR is involved in regulation of the dltABCD operon, the gene products of which add the d-Ala on wall teichoic acid (WTA). Investigation of WTA isolated from the S. aureus RN6390 ΔgraR strain by NMR spectroscopy showed a clear negative effect that graR deletion has on the d-Ala content of WTA. Moreover, complementation of ΔgraR mutant with graR lacking the Stk1 phosphorylation sites mirrors this effect. These findings provide evidence that GraR is a target of Stk1 in vivo and suggest that modification of WTA by d-Ala is modulated by Stk1. The crosstalk between these two otherwise independent signaling pathways may facilitate S. aureus interaction with its environment to modulate processes such as cell growth and division and virulence.
SummaryThe influence of sample preparation on ion suppression is studied using the model compounds ropivacaine and 3OH-ropivacaine. Three sample preparation techniques are assessed: Solid Phase Extraction (SPE), Direct Dilution and Protein Precipitation.Although requiring more method development and being more time-consuming than the k, vo alternative preparation techniques, SPE had a greater impact on reducing ion suppression.
Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.
Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.