Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine β-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 β subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis
Use of the restriction enzyme to treat diseases:
There is substantial evidence of impaired DNA repair activities in Alzheimer’s disease (AD) neurons and peripheral tissues, inducing some investigators to speculate that this could partially result from promoter hypermethylation of DNA repair genes, resulting in gene silencing in those tissues. In the present study a screening cohort composed by late-onset AD (LOAD) patients and healthy matched controls was evaluated with a commercially available DNA methylation array for the assessment of the methylation levels of a panel of 22 genes involved in major DNA repair pathways in blood DNA. We then applied a cost-effective PCR based methylation-sensitive high-resolution melting (MS-HRM) technique, in order to evaluate the promoter methylation levels of the following DNA repair genes: OGG1, PARP1, MRE11A, BRCA1, MLH1, and MGMT. The analysis was performed in blood DNA from 56 LOAD patients and 55 matched controls, including the samples previously assessed with the DNA methylation array as validating samples. Both approaches revealed that all the investigated genes were largely hypomethylated in LOAD and control blood DNA, and no difference between groups was observed. Collectively, present data do not support an increased promoter methylation of some of the major DNA repair genes in blood DNA of AD patients.
In the early 1950s, experiments by two teams of researchers, Salvador Luria working with Mary Human and Joe Bertani working with Jean Weigle, showed that some strains of bacteria were more resistant to viral infections than others. Viruses that infect bacterial cells are called bacteriophages. Their main goal is to produce more bacteriophages by injecting their genome into a bacterial host cell, using the host cell machinery to copy their genome, and expressing bacteriophage genes. The researchers found, however, that some strains of bacteria appeared to be less vulnerable to bacteriophage infections than others and resisted the hijacking of their cell machinery by bacteriophages. A deeper look into the apparent self-defense mechanisms of these bacteriophage-resistant bacteria revealed their secret weapon: a group of enzymes called restriction endonucleases, or restriction enzymes. These enzymes opened the path to a powerful research tool that scientists later used not only to sequence genomes, but also to create the first synthetic cell, two scientific research milestones that affect us all in some way.