A Collaborative Study: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B, B, G, and G; deoxynivalenol; fumonisins B, B, and B; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 C uniformly labeled mycotoxins (C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.

Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms.

The impetus for discovery and evaluation of protein biomarkers has been accelerated by recent development of advanced technologies for rapid and broad proteome analyses. Mass spectrometry (MS)-based protein assays hold great potential for in vitro biomarker studies. Described here is the development of a multiplex mass spectrometric immunoassay (MSIA) for quantification of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apoC-II), apolipoprotein C-III (apoC-III) and their proteoforms.

Elevated plasma albumin and apolipoprotein A-I oxidation under suboptimal specimen storage conditions.

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC-electrospray ionization-MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at -80 °C, -20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at -20 °C.

Parallel workflow for high-throughput (>1,000 samples/day) quantitative analysis of human insulin-like growth factor 1 using mass spectrometric immunoassay.

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories.

Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: potential applications to cardiovascular disease and diabetes.

Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)--the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients.

Building multidimensional biomarker views of type 2 diabetes on the basis of protein microheterogeneity.

Background: In 2008, the US Food and Drug Administration (FDA) issued a Guidance for Industry statement formally recognizing (during drug development) the conjoined nature of type 2 diabetes (T2D) and cardiovascular disease (CVD), which has precipitated an urgent need for panels of markers (and means of analysis) that are able to differentiate subtypes of CVD in the context of T2D. Here, we explore the possibility of creating such panels using the working hypothesis that proteins, in addition to carrying time-cumulative marks of hyperglycemia (e.g.

Structure and function of YghU, a nu-class glutathione transferase related to YfcG from Escherichia coli.

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.

Evolution of the antibiotic resistance protein, FosA, is linked to a catalytically promiscuous progenitor.

The fosfomycin (1) resistance proteins FosA and FosX in pathogenic microorganisms are related to a catalytically promiscuous progenitor encoded in a phn operon in Mesorhizobium loti. The mlr3345 gene product (FosX(Ml)) from M. loti has a very low epoxide hydrolase activity and even lower glutathione transferase activity toward 1 and does not confer resistance to the antibiotic.

Structure and mechanism of the genomically encoded fosfomycin resistance protein, FosX, from Listeria monocytogenes.

The fosfomycin resistance protein, FosX, catalyzes the hydration of the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. Genes encoding the enzyme are found in several pathogenic microorganisms. The structure and mechanism of action of the genomically encoded FosX enzyme from Listeria monocytogenes (FosXLMATCC) obtained from the American Type Culture Collection are reported.

Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis.

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals.

Evidence for a new metal in a known active site: purification and characterization of an iron-containing quercetin 2,3-dioxygenase from Bacillus subtilis.

The protein YxaG from Bacillus subtilis, of previously unknown function, was found to have quercetin 2,3-dioxygenase activity when overexpressed in Escherichia coli. The enzyme converts the flavonol quercetin to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide, indicating that it performs the same reaction and yields the same products as the well-characterized copper-containing quercetin 2,3-dioxygenase from Aspergillus. In contrast to the Aspergillus protein, YxaG contains iron, and the enzyme is sensitive to strong Fe(II) chelators, similar to the extensively studied catechol dioxygenases.