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Although sequencing a single human genome was a monumental effort a decade ago, more than one thousand genomes have now been sequenced. The task ahead lies in transforming this information into personalized treatment strategies that are tailored to the unique genetics of each individual. One important aspect of personalized medicine is patient-to-patient variation in drug response. Pharmacogenomics addresses this issue by seeking to identify genetic contributors to human variation in drug efficacy and toxicity. Here, we present a summary of the current status of this field, which has evolved from studies of single candidate genes to comprehensive genome-wide analyses. Additionally, we discuss the major challenges in translating this knowledge into a systems-level understanding of drug physiology with the ultimate goal of developing more effective personalized clinical treatment strategies.

The focus of this study was on the assessment of technology that might be of clinical utility in identification, quantification, characterization of carbonylation in human plasma proteins. Carbonylation is widely associated with oxidative stress diseases. Breast cancer patient samples were chosen as a stress positive case based on the fact that oxidative stress has been reported to be elevated in this disease. Measurements of 8-isoprostane in plasma confirmed that breast cancer patients in this study were indeed experiencing significant oxidative stress. Carbonyl groups in proteins from freshly drawn blood were derivatized with biotin hydrazide after which the samples were dialyzed and the biotinylated proteins subsequently selected, digested and labeled with iTRAQ™ heavy isotope coding reagent(s). Four hundred sixty proteins were identified and quantified, 95 of which changed 1.5 fold or more in concentration. Beyond confirming the utility of the analytical method, association of protein carbonylation was examined as well. Nearly one fourth of the selected proteins were of cytoplasmic, nuclear, or membrane origin. Analysis of the data by unbiased knowledge assembly methods indicated the most likely disease associated with the proteins was breast neoplasm. Pathway analysis showed the proteins which changed in carbonylation were strongly associated with Brca1, the breast cancer type-1 susceptibility protein. Pathway analysis indicated the major molecular functions of these proteins are defense, immunity and nucleic acid binding.

Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post-translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the alpha-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and a wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing the identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation.

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi-PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC); fractions from the RPC column were tryptic digested, and the tryptic digest was fractionated by RPC before being identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.

Background Tacrolimus is an immunosuppressive drug that is used to lower the activity of the patient’s immune system to prevent organ rejection. Unfortunately, there is limited data regarding the therapeutic equivalency of generic tacrolimus formulations especially in children. We report the case of a pediatric patient having an inability to achieve a therapeutic trough level for tacrolimus after conversion from brand name to the generic formulation. Case presentation A 17-month-old male patient diagnosed with T-cell acute lymphoblastic leukemia underwent allogeneic stem cell transplantation. The patient initially received intravenous (IV) tacrolimus for graft-versus-host disease (GVHD) prophylaxis and achieved therapeutic levels. The patient was then switched to an oral brand formulation of tacrolimus, and was able to maintain trough levels within the therapeutic range. After being discharged, the patient received the generic formulation of tacrolimus from an outside pharmacy and the care team was unable to reach therapeutic levels despite multiple dose escalations. Returning to brand name tacrolimus resulted in prompt achievement of therapeutic levels. Conclusions A likely etiology for the inability to achieve therapeutic trough levels in this patient is the change in formulation from brand formulation to generic version. Other factors including drug-drug interaction, preparation of the medication by a different pharmacy, drug-food interaction and genetic factors were also considered. Physicians and pharmacists must be aware of the inability to achieve targeted therapeutic concentrations of tacrolimus resulting from the conversion of brand name to the generic formulation until these generic formulations are tested in clinical trials in a pediatric population.