Integration Of ASP-Specific Microwave-Accelerated Acid Hydrolysis into Proteomic Analyses

Presented in this work is a novel "bottom up" proteomics approach to protein identification and characterization that utilizes microwave-accelerated acid hydrolysis combined with mass spectrometric methods and bioinformatics. Results of this study demonstrate that this strategy is a robust alternative to residue specific enzymatic cleavage methods for generating peptides. Cleavage of proteins was shown to be site-specific at aspartate, as evaluated by matrix-assisted laser desorption ionization time of flight mass spectrometry and liquid chromatography tandem mass spectrometry. A bioinformatic analysis indicates that proteins will be cleaved at Asp to provide peptides that are longer than tryptic peptides, and which contain more basic residues, on average. The feasibility of this digestion method was first demonstrated on a pure protein standard, ovalbumin, and further developed for applications in rapid microorganism identification, and whole organelle processing. Digestion of ovalbumin and analysis by mass spectrometry provided ~80% sequence coverage. Bacillus spores, the RNA virus, bacteriophage MS2, and the DNA virus, human adenovirus type 5, were identifiable, based on the analysis digestion products generated by rapid digestion of protein biomarkers released by acid. Whole ribosomes isolated from Saccharomyces cervasiae were processed directly to peptides, which enabled identification of 58 of 79 ribosomal proteins by LC tandem mass spectrometry. Finally, this digestion method was shown to be compatible with a proteolytic 18O labeling strategy, which enables rapid relative quantitation of proteins

Identification of β-Lactamase in Antibiotic-Resistant Bacillus cereus Spores▿

β-Lactamase type I is reported for the first time to occur in the sporulated form in a penicillin-resistant Bacillus species. The enzyme was readily characterized from the B. cereus 5/B line (ATCC 13061) by mass spectrometry and two-dimensional gel electrophoresis

SOX10 post-translational modifications identified in MG132-treated 501mel cells.

<p>This schematic representing the SOX10 protein indicates known domains, SOXE conserved regions, and phosphorylated residues, as follows: black bars show known phosphorylation sites, green bars show known sites that were confirmed in this study, and red bars show novel sites from this study. The phosphorylated residues S224, S232, T240 and T244 were observed on numerous peptide fragments, and one or all four are plausible; their close proximity and the limited fragmentation capability in the digest restrict more precise determination among these residues. The nuclear localization and nuclear export signal regions are unaffected by the phosphorylation sites.</p

Mutation of SOX10 phosphorylation sites causes distinct changes in protein stability.

<p>Cycloheximide pulse-chase assays in 501mel (A-C) and MeWo (D-F) cells revealed altered stability of SOX10 phospho-mutants compared to WT SOX10 protein. A. WT SOX10 showed a half-life of 8.3 hours in 501mel cells. B,C. Stability of SOX10 phospho-mutants S24A and T240A is not significantly different from WT SOX10 in 501mel cells (two-way ANOVA, p = 0.25). D. WT SOX10 stabilty in MeWo cells exhibited a half-life of 19.5 hours. E. S24A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 4.7 hours. F. T240A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 11.7 hours. Both the S24A and the T240A mutant proteins exhibited significant differences relative to WT SOX10 protein in MeWo cells (two-way ANOVA, p = 0.0057 for protein type, p<0.0001 for time and interaction); by Bonferroni’s multiple comparisons post-test, these differences were significant for SOX10 S24A from 4 hours through 10 hours, and were significant for SOX10 T240A from 4 hours through 16 hours (P-values: *≤0.05, **≤0.01, ***≤0.001, ***≤0.0001). Data are compiled from 3 independent assays, with standard deviations plotted.</p