\u3ci\u3eIn Vitro\u3c/i\u3e Stable Isotope Labeling for Discovery of Novel Metabolites by Liquid Chromatography-Mass Spectrometry: Confirmation of γ-Tocopherol Metabolism in Human A549 Cell

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on metabolism of γ-tocopherol in human A549 cell. Method is based on LC-MS analysis of in vitro stable isotope labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC-MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by identification of products of γ-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and Nmethyl- d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four γ-tocopherol metabolites were identified including 9\u27-COOH, 11\u27-COOH, 13\u27-COOH and 13\u27-OH. In addition, the developed LC-MS method can also be used for the fast and sensitive quantitative analysis of γ-tocopherol and other forms of vitamin E related compounds

Influence of dietary constituents on intestinal absorption of aluminum

Orally-ingested aluminum compounds have been implicated in the development of dialysis encephalopathy, osteomalacic dialysis osteodystrophy and other disorders in both hemodialyzed and nonhemodialyzed patients suffering from chronic renal failure [1–10]. Both dialysate aluminum content [7, 11, 12] and aluminum-containing phosphate binding agents [12–15] have been identified as contributing to hyperaluminemia in uremic patients. The health threat from dialysate fluids has been reduced by the recommendation that the dialysate contains less than 10 µg/liter of aluminum [16]. Alternative phosphate-binding agents which do not contain aluminum are available but these agents are not free of problems [17], and uremic patients continue to ingest significant doses of aluminum-containing phosphate binding agents.Aluminum is the most common metal in the biosphere of humans but, aside from uremic patients, causes no widespread toxicity. This may be as a result of the extremely limited solubility of aluminum at the pH range of the small intestine and blood [18]. Advances in analytical chemistry have made it possible to measure picogram quantities of aluminum in body fluids, thus enabling accurate determination of plasma aluminum levels in the part per billion (µg/liter) range. These analytical techniques have shown that orally ingested aluminum-containing antacids elevate plasma aluminum levels in man [13]. Balance studies monitoring aluminum absorption and elimination revealed an average positive balance from 23 to 313mg of aluminum per day when diets were supplemented with 1 to 3g of aluminum per day [15]. These studies show that a small fraction of the ingested aluminum is absorbed. This absorption presents potential toxic effects to uremic patients whose ability to eliminate aluminum is impaired.In addition, Slanina et al [19] have shown that addition of citric acid to aluminum-supplemented dietary regimens results in blood aluminum levels that are significantly higher than those found in subjects treated with aluminum-supplemented dietary regimens alone. This result suggests that dietary factors may contribute to aluminum absorption.This study was undertaken to determine if the form of aluminum present in the intestinal lumen significantly affects the absorption of aluminum following oral ingestion

An Automated Method for the Analysis of Stable Isotope Labeling Data in Proteomics

An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios

Proteomic analysis of Arabidopsis glutathione S-transferases from benoxacor- and copper-treated seedlings

Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions

Patterned Protein Microarrays for Bacterial Detection

Abstract Patterned microarrays of antibodies were fabricated and tested for their ability to bind targeted bacteria. These arrays were used in a series of bacterial immunoassays to detect E. coli 0157:H7 and Renibacterium Salmoninarum (RS). Microarrays were fabricated using microcontact printing (µCP) and characterized using scanning probe microscopy (SPM). The high-resolution SPM imaging showed that targeted bacteria had a higher binding selectivity to complementary antibody patterns than to unfunctionalized regions of the substrate. Additional studies indicated a significant reduction in binding of bacteria when the microarrays were exposed to non-specific bacteria. These studies demonstrate how protein microarrays could be developed into useful platforms for sensing microorganisms.

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals

Anthropogenic perturbation of the carbon fluxes from land to ocean

A substantial amount of the atmospheric carbon taken up on land through photosynthesis and chemical weathering is transported laterally along the aquatic continuum from upland terrestrial ecosystems to the ocean. So far, global carbon budget estimates have implicitly assumed that the transformation and lateral transport of carbon along this aquatic continuum has remained unchanged since pre-industrial times. A synthesis of published work reveals the magnitude of present-day lateral carbon fluxes from land to ocean, and the extent to which human activities have altered these fluxes. We show that anthropogenic perturbation may have increased the flux of carbon to inland waters by as much as 1.0 Pg C yr-1 since pre-industrial times, mainly owing to enhanced carbon export from soils. Most of this additional carbon input to upstream rivers is either emitted back to the atmosphere as carbon dioxide (~0.4 Pg C yr-1) or sequestered in sediments (~0.5 Pg C yr-1) along the continuum of freshwater bodies, estuaries and coastal waters, leaving only a perturbation carbon input of ~0.1 Pg C yr-1 to the open ocean. According to our analysis, terrestrial ecosystems store ~0.9 Pg C yr-1 at present, which is in agreement with results from forest inventories but significantly differs from the figure of 1.5 Pg C yr-1 previously estimated when ignoring changes in lateral carbon fluxes. We suggest that carbon fluxes along the land–ocean aquatic continuum need to be included in global carbon dioxide budgets.Peer reviewe