Tandem Mass Spectrometry-based Method Research Articles

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening.

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of Δ1 -piperideine-6-carboxylate and α-aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4 months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques.

Multi-mycotoxin screening of feed and feed raw materials from Africa

As animal feed is prone to infestation with mycotoxin-producing fungi, mycotoxin contamination of feed should be monitored. Here, we report a multi-mycotoxin survey of feed samples from Africa. We determined the concentrations of aflatoxins, fumonisins, deoxynivalenol, T-2 toxin, zearalenone and ochratoxin A in 1,045 samples of finished feed and feed raw materials (maize, maize silage, other cereals, etc.) from South Africa and 318 samples from Algeria, Tunisia, Morocco, Senegal, Côte d’Ivoire, Nigeria, Ghana, Namibia, Uganda, Kenya, Tanzania, Zambia and Madagascar. We compared the measured mycotoxin concentrations to regulatory limits or guidance values that are in effect in the European Union and analysed the co-occurrence of these mycotoxins. To determine the occurrence of other fungal secondary metabolites, a subset of the samples was analysed using a multi-analyte liquid chromatography tandem mass spectrometry-based method for the simultaneous detection of over 700 fungal metabolites. We found that 33.3% of maize samples and 54.4% of finished feed samples from Senegal, Côte d’Ivoire, Nigeria, Ghana, Namibia, Uganda, Kenya and Tanzania exceeded the European regulatory limit of 20 ng/g aflatoxins. TheFusarium mycotoxins zearalenone, fumonisins and deoxynivalenol were prevalent in all commodities from all countries, but concentrations were in most cases below European guidance values. Concentrations of deoxynivalenol and zearalenone were correlated. Several otherFusarium metabolites occurred frequently (e.g. moniliformin, beauvericin, aurofusarin) or in high concentrations (e.g. aurofusarin, fusaproliferin). Furthermore, high levels of diplodiatoxin were occasionally detected in samples from South Africa and theAlternaria metabolite tenuazonic acid was prevalent and reached high concentrations. In conclusion, aflatoxins frequently occurred in African feed samples in potentially unsafe concentrations. WhileFusarium mycotoxins mostly occurred in concentrations below European guidance values, a correlation between deoxynivalenol and zearalenone concentrations suggests that toxicological interactions of these compounds deserve attention. Several less investigated fungal secondary metabolites occurred frequently or reached high concentrations.

Simultaneous detection of the tetrachloroethylene metabolites S-(1,2,2-trichlorovinyl) glutathione, S-(1,2,2-trichlorovinyl)-L-cysteine, and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine in multiple mouse tissues via ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry

ABSTRACTTetrachloroethylene (perchloroethylene; PERC) is a high-production volume chemical and ubiquitous environmental contaminant that is hazardous to human health. Toxicity attributed to PERC is mediated through oxidative and glutathione (GSH) conjugation metabolites. The conjugation of PERC by glutathione-s-transferase to generate S-(1,2,2-trichlorovinyl) glutathione (TCVG), which is subsequently metabolized to form S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) is of special importance to human health. Specifically, TCVC may be metabolized to N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (NAcTCVC) which is excreted through urine, or to electrophilic metabolites that are nephrotoxic and mutagenic. Little is known regarding toxicokinetics of TCVG, TCVC, and NAcTCVC as analytical methods for simultaneous determination of these metabolites in tissues have not yet been reported. Hence, an ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry-based method was developed for analysis of TCVG, TCVC, and NAcTCVC in liver, kidneys, serum, and urine. The method is rapid, sensitive, robust, and selective for detection all three analytes in every tissue examined, with limits of detection (LOD) ranging from 1.8 to 68.2 femtomoles on column, depending on the analyte and tissue matrix. This method was applied to quantify levels of TCVG, TCVC, and NAcTCVC in tissues from mice treated with PERC (10 to 1000 mg/kg, orally) with limits of quantitation (LOQ) of 1–2.5 pmol/g in liver, 1–10 pmol/g in kidney, 1–2.5 pmol/ml in serum, and 2.5–5 pmol/ml in urine. This method is useful for further characterization of the GSH conjugative pathway of PERC in vivo and improved understanding of PERC toxicity.

Analysis of Salmonella PhoP/PhoQ regulation by dimethyl-SRM-based quantitative proteomics

SRM (selected reaction monitoring), a tandem mass spectrometry-based method characterized by high repeatability and accuracy, is an effective tool for the quantification of predetermined proteins. In this study, we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run. By applying this method to the Salmonella PhoP/PhoQ two-component system, we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg2+ concentrations could be divided into two distinct patterns. For the time-course SRM experiment, we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns, providing a new clue regarding PhoP/PhoQ activation and regulation. Moreover, the results for iron homeostasis proteins in response to Mg2+ concentrations revealed that the PhoP/PhoQ two-component system may serve as a repressor for iron uptake proteins. And ribosomal protein levels clearly showed a response to different Mg2+ concentrations and to time.

Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated fatty acid supplementation by a highly specific liquid chromatography-tandem mass spectrometry-based method.

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) have beneficial effects in many pathological processes, especially cardiovascular disease, and their protective eicosanoid metabolites are thought to play important roles. However, how ω-3 PUFAs affect the eicosanoid profile has not been elucidated comprehensively. Here, we systematically analyzed the eicosanoid metabolites induced by ω-3 PUFA supplementation. We developed an LC-MS/MS-based method covering 32 arachidonic acid (ARA) metabolites and 37 ω-3 PUFA-derived products. The limits of detection for eicosanoids were between 0.0625 and 1 pg and the detection specificity was optimized. We then quantified eicosanoids in mouse and human plasma and mouse aorta samples after ω-3 PUFA supplementation. Levels of EPA hydroxyl products, 4-HDoHE, 17,18-EEQ, 17,18-DiHETE, TXB2, and LXA4 were significantly changed in both mouse samples, and those of 2-series PGs, EDPs and DHA hydroxyl products were changed in aorta samples. Correlation network analysis of mouse plasma data revealed that some eicosanoids had higher connection degree or betweenness centrality score than others after ω-3 PUFA supplementation. Eicosanoids in human plasma were profiled across five time points after ω-3 PUFA supplementation. Fuzzy c-mean clustering algorithm suggested that the time curves of eicosanoid activity could be described with three kinetic patterns: sustained upregulation, short-term upregulation, and downregulation. This is the first systematic profiling of eicosanoids with ω-3 PUFA supplementation. The highly specific eicosanoid metabolomic and related data analysis methods would be powerful tools for comprehensive eicosanoid study.

A tandem mass spectrometry-based method to assess the architectural purity of synthetic polymers: a case of a cyclic polylactide obtained by click chemistry

A tandem mass spectrometry-based method is developed to determine the degree of purity achieved in the cyclization of a linear poly(l-lactide).

Quantitative Proteomic Approach for MicroRNA Target Prediction Based on 18O/16O Labeling.

MOTIVATIONAmong many large-scale proteomic quantification methods, 18O/16O labeling requires neither specific amino acid in peptides nor label incorporation through several cell cycles, as in metabolic labeling; it does not cause significant elution time shifts between heavy- and light-labeled peptides, and its dynamic range of quantification is larger than that of tandem mass spectrometry-based quantification methods. These properties offer 18O/16O labeling the maximum flexibility in application. However, 18O/16O labeling introduces large quantification variations due to varying labeling efficiency. There lacks a processing pipeline that warrants the reliable identification of differentially expressed proteins (DEPs). This motivates us to develop a quantitative proteomic approach based on 18O/16O labeling and apply it on Kaposi sarcoma-associated herpesvirus (KSHV) microRNA (miR) target prediction. KSHV is a human pathogenic γ-herpesvirus strongly associated with the development of B-cell proliferative disorders, including primary effusion lymphoma. Recent studies suggest that miRs have evolved a highly complex network of interactions with the cellular and viral transcriptomes, and relatively few KSHV miR targets have been characterized at the functional level. While the new miR target prediction method, photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP), allows the identification of thousands of miR targets, the link between miRs and their targets still cannot be determined. We propose to apply the developed proteomic approach to establish such links.METHODWe integrate several 18O/16O data processing algorithms that we published recently and identify the messenger RNAs of downregulated proteins as potential targets in KSHV miR-transfected human embryonic kidney 293T cells. Various statistical tests are employed for picking DEPs, and we select the best test by examining the enrichment of PAR-CLIP-reported targets with seed match to the miRs of interest among top ranked DEPs returned by statistical tests. Subsequently, the list of DEPs picked by the selected statistical test is filtered with the criteria that they must have downregulated gene expressions, must have reported as targets by an miR target prediction algorithm SVMcrio, and must have reported as targets by PAR-CLIP.RESULTWe test the developed approach in the problem of finding targets of KSHV miR-K1. The RNAs of three DEPs are identified as miR-K1 targets, among which RAB23 and HNRNPU are novel. Results from both Western blotting and Luciferase reporter assays confirm the novel targets. These results show that the developed quantitative approach based on 18O/16O labeling can be combined with genomic, PAR-CLIP, and target prediction algorithms for the confident identification of KSHV miR targets. The developed approach could also be applied in other applications.

Multiresidue analysis of over 200 pesticides in cereals using a QuEChERS and gas chromatography–tandem mass spectrometry-based method

A simple and high-throughput multiresidue pesticide analysis method was developed and validated for 219 pesticides in cereals (corn, wheat flour and rice) based on QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure combined with gas chromatography–triple quadrupole mass spectrometry (GC–MS/MS). Different buffer system (acetate- and citrate-buffered) and sample to solvent ratios (sample amount) were compared in the modified QuEChERS procedure to get better recovery and clean-up results. The limits of quantification (LOQ) ranged between 5 and 50μgkg−1, and for the majority of the pesticides the LOQ were 5μgkg−1, which were below the regulatory maximum residue limits. The coefficient of determination (r2) was >0.99 within the calibration linearity range of 2–200μgkg−1 for the majority of the pesticides. Most recoveries at 5, 10, 20, 50, 100 and 200μgkg−1 were in the range 70–120% (n=6) with associated RSDs<20% indicating satisfactory accuracy.

Identification of specific corrinoids reveals corrinoid modification in dechlorinating microbial communities.

Cobalamin and other corrinoids are essential cofactors for many organisms. The majority of microbes with corrinoid-dependent enzymes do not produce corrinoids de novo, and instead must acquire corrinoids produced by other organisms in their environment. However, the profile of corrinoids produced in corrinoid-dependent microbial communities, as well as the exchange and modification of corrinoids among community members have not been well studied. In this study, we applied a newly developed liquid chromatography tandem mass spectrometry-based corrinoid detection method to examine relationships among corrinoids, their lower ligand bases and specific microbial groups in microbial communities containing Dehalococcoides mccartyi that has an obligate requirement for benzimidazole-containing corrinoids for trichloroethene respiration. We found that p-cresolylcobamide ([p-Cre]Cba) and cobalamin were the most abundant corrinoids in the communities. It suggests that members of the family Veillonellaceae are associated with the production of [p-Cre]Cba. The decrease of supernatant-associated [p-Cre]Cba and the increase of biomass-associated cobalamin were correlated with the growth of D. mccartyi by dechlorination. This supports the hypothesis that D. mccartyi is capable of fulfilling its corrinoid requirements in a community through corrinoid remodelling, in this case, by importing extracellular [p-Cre]Cba and 5,6-dimethylbenzimidazole (DMB) (the lower ligand of cobalamin), to produce cobalamin as a cofactor for dechlorination. This study also highlights the role of DMB, the lower ligand produced in all of the studied communities, in corrinoid remodelling. These findings provide novel insights on roles played by different phylogenetic groups in corrinoid production and corrinoid exchange within microbial communities. This study may also have implications for optimizing chlorinated solvent bioremediation.

Screening of Lysosomal Storage Disorders: Application of the Online Trapping-and-Cleanup Liquid Chromatography/Mass Spectrometry Method for Mucopolysaccharidosis I

In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.

Fate of triclosan in tertiary wastewater treatment: chlorination

Behavior of the antimicrobial triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) was investigated under laboratory chlorination conditions and in a wastewater treatment plant discharging 380 million liters daily to the Delaware River, USA. Reactions of triclosan with chlorine were investigated using concentrations and exposure time typical of municipal wastewater treatment plants, i.e., 1 h contact time and average 1–2 mg/L residual chlorine. In reagent water, triclosan reacted quickly, transforming into mono- and dichlorinated species and further into dichlorophenol and trichlorophenol. However, triclosan remained stable for up to 2 h in wastewater samples chlorinated under these conditions. To confirm observed behavior under field conditions, a liquid chromatography tandem mass spectrometry-based analytical method capable of monitoring triclosan and its transformation products in wastewater was developed. Qualitative and quantitative wastewater characterization before and after chlorination are presented. Triclosan was present at the same concentration (P &amp;gt; 0.05) in pre-chlorination and post-chlorination aqueous wastewater samples (mean 368 ng/L). This finding is consistent with the non-detection of specific triclosan transformation products above sample reporting limits (30.0–100 ng/L), but contrasts markedly with detection of chlorination transformation products reported in reagent water. These data suggest the importance of influent matrix components in chlorination reactions of triclosan in contaminated wastewater under treatment plant conditions.

Rapid liquid chromatography–tandem mass spectrometry-based method for the analysis of alcohol ethoxylates and alkylphenol ethoxylates in environmental samples

A sensitive and selective method for the determination of alcohol ethoxylates (AEOs) and alkylphenol ethoxylates (APEOs) using solid-phase extraction (SPE) and LC–MS/MS was developed and applied to the analysis of water samples. All AEO and APEO homologues, a total of 152 analytes, were analyzed within a run time of 11min, and the MS allowed for the detection of ethoxymers containing 2–20 ethoxy units (nEO=2–20). The limits of detection (LOD) were as low as 0.1pg injected, which generally increased as nEO increased (e.g., as high as 300pg for nEO=20). Additionally, the responses of the various ethoxymers varied by orders of magnitude, with ethoxymers with nEO=3–5 being the most sensitive and those with nEO>15 producing the least response in the MS. Absolute extraction recoveries of the analytes ranged from 37% to 69% in ultrapure water (RSD≤20%), with the recovery depending on the length of the alkyl chain. Abiotic stability studies were performed, and C14–18 ethoxylates showed significant degrees of degradation. Water samples from the Colorado River were then analyzed for AEOs and APEOs, with absolute extraction recoveries ranging from 33% to 45% (RSD≤12%). The predominant species observed in most samples were the octylphenol (OP) and nonylphenol (NP) ethoxylates, which contained total concentrations that were greater than 100ng/L APEOs in a couple samples. Other AEO homologues were identified in the river water samples, including C13, C15, C16, and C18 ethoxylates, but these compounds were generally present at much lower levels (i.e., <50ng/L total concentration).

Relationship of serum estrogens and estrogen metabolites to postmenopausal breast cancer risk: a nested case-control study

IntroductionElevated levels of circulating estrogens are linked to breast cancer risk among postmenopausal women but little is known about the importance of estrogen metabolism. A recently developed liquid chromatography tandem mass spectrometry-based method (LC-MS/MS) measuring a panel of 15 estrogen metabolites (EM) has been evaluated in one study, linking high levels of 2-pathway metabolites relative to the parent estrogens to reduced breast cancer risk. We analyzed this panel of EM in a nested case-control study of postmenopausal breast cancer.MethodsBetween 1977 and 1987, 6,915 women provided blood samples to the Columbia Missouri Serum Bank and were followed for incident breast cancer through December 2002. We studied 215 postmenopausal breast cancer cases and 215 matched controls who were postmenopausal and not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway (hydroxylation at the C-2, C-4 or C-16 positions of the steroid ring) and by ratios of the groupings. Logistic regression models controlling for matching and breast cancer risk factors were used to calculate quartile-specific odds ratios (ORs) and 95% CIs.ResultsSignificant elevated risks were not observed for individual EM, except for quartiles of 16-epiestriol (P trend = 0.07). The OR for total EM, the parent estrogens estrone and estradiol, and 2-pathway catechol EM (2-hydroxyestrone and 2-hydroxyestradiol) were elevated but the trends were not statistically significant. Among 2-pathway metabolites, risks for the highest levels of 2-hydroxyestrone-3-methyl ether and 2-methoxyestradiol were reduced; ORs for women in the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.53 (95% CI = 0.30 to 0.96), respectively. Overall, women with higher levels of 2-pathway EM had a reduced risk of breast cancer, which remained after accounting for levels of parent EM, 4-pathway EM and 16-pathway EM (all trends, P <0.11).ConclusionsWomen with more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer. Further studies are needed to clarify the risks for specific EM and complex patterns of estrogen metabolism. This will require aggregation of EM results from several studies.

A rapid liquid chromatography tandem mass spectrometry-based method for measuring propranolol on dried blood spots

Propranolol, a non-selective beta blocker drug, is used in young infants and newborns for treating several heart diseases; its pharmacokinetics has been extensively evaluated in adult patients using extrapolation to treat pediatric population.The purpose of the present study was to develop and validate a method to measure propranolol levels in dried blood spots. The analysis was performed by using liquid chromatography/tandem mass spectrometry operating in multiple reaction monitoring mode. The calibration curve in matrix was linear in the concentration range of 2.5–200μg/L with correlation coefficient r=0.9996. Intra-day and inter-day precisions and biases were less than 8.0% (n=10) and 11.5% (n=10) respectively. The recoveries ranged from 94 to 100% and the matrix effect did not result in a severe signal suppression.Propranolol on dried blood spot showed a good stability at three different temperatures for one month.This paper describes a micromethod for measuring propranolol levels on dried blood spot, which determines a great advantage in neonates or young infants during pharmacokinetic studies because of less invasive sampling and small blood volume required.

Plasma corticosteroid profiling: brief opinion of its current status in clinical diagnosis and research

Adrenal steroid biosynthesis comprises a series of dynamically interrelated, enzyme-catalyzed reactions in two separate compartments, the zona glomerulosa and the zona fasciculata/reticularis. End products (cortisol, aldosterone and androgens), together with a proportion of the intermediate compounds, appear in the circulation as a characteristic profile. Rare deficiencies of individual enzymes modify this profile in a recognizable way. Previous exhaustive profiling suggests that their diagnosis can now often be made on the basis of single-compound analyses with concomitant genetic tests. However, high-capacity liquid chromatography coupled with tandem mass spectrometry-based methods are facilitating profiling of large population samples, revealing that smaller differences in enzyme efficiency, indicated by potentially more complex corticosteroid patterns, may be related to clinical wellbeing in a much larger proportion of the population.

New Strategy for the Screening of Lysosomal Storage Disorders: The Use of the Online Trapping-and-Cleanup Liquid Chromatography/Mass Spectrometry

The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.

Measurement of carnitine biosynthesis enzyme activities by tandem mass spectrometry: Differences between the mouse and the rat

Although the mouse frequently is used to study metabolism and deficiencies therein, little is known about carnitine biosynthesis in this animal. To this point, only laborious procedures have been described to measure the activity of carnitine biosynthesis enzymes using subcellular fractions as the enzyme source. We developed two simple tandem mass spectrometry-based methods to determine the activity of three carnitine biosynthesis enzymes (6- N-trimethyllysine dioxygenase, 4-trimethylaminobutyraldehyde dehydrogenase, and 4-trimethylaminobutyric acid dioxygenase) in total homogenates that can be prepared from frozen tissue. The new assays were used to characterize these enzymes in mouse liver homogenate. Because carnitine biosynthesis has been studied extensively in the rat, we compared the mouse tissue distribution of carnitine biosynthesis enzyme activities and levels of the biosynthesis metabolites with those in the rat to determine which tissues contribute to carnitine biosynthesis in these species. Surprisingly, large differences in enzyme activities were found between the rat and the mouse, whereas carnitine biosynthesis metabolite levels were very similar in both species, possibly due to the different kinetic properties of the first enzyme of carnitine biosynthesis. Also, muscle carnitine levels were found to vary considerably between these two species, suggesting that there is a metabolic dissimilarity between the mouse and the rat.

Identification and Characterization of a Novel p300-mediated p53 Acetylation Site, Lysine 305

Post-translational modifications serve as important regulatory elements in modulating the transcriptional activity of the tumor suppressor protein p53. We have previously reported a tandem mass spectrometry-based method (viz. selected ion tracing analysis) that can be applied to the identification of phosphopeptides as well as exact mapping of the phosphorylated residues within. In this study, we describe the application of the same strategy for the identification of p300 acetyltransferase-mediated acetylation sites on p53. Consistent with the previous finding, lysines 370, 372, 373, 381, and 382 were detected by this modified selected ion tracing method as the target sites of p300 in vitro. Moreover, two novel acetylation sites, Lys-292 and Lys-305, were also found. Immunoblotting using anti-acetyl-Lys-305 antibody confirmed this discovery and demonstrated that Lys-305 could be acetylated by p300 both in vitro and in vivo. We also show that an alanine or glutamine substitution at Lys-305 (K305A or K305Q) suppressed the transcriptional activity of p53, whereas an arginine mutation (K305R) increased the transcriptional activity. Thus, p300 may further regulate the transcriptional activity of p53 through a novel acetylation site, Lys-305.