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Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98-109 % were achieved for each ceramide. Addition of corresponding D-7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC-MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case-control study. Thus, our LC-MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography-high-resolution mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5-7.7 % and within-lab reproducibility (RSD(WLR)) of 17 %. The method was used for analysis of100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops.

The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates ( reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)-mass spectrometry ( MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e. g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates ( reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)-mass spectrometry ( MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e. g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.

Burned greasy deposits were found inside shells of the large Nile bivalve Chambardia rubens, excavated in an eight- to tenth- century AD church of the Coptic monastery of Bawit, Egypt, and supposedly used as oil lamps. The residues were subjected to a combination of chromatographic residue analysis techniques. The rather high concentrations of unsaturated fatty acids, as analysed by gas chromatography (GC) in the methylated extract, suggest the presence of a vegetal oil. Analysis of the stable carbon isotopes (delta C-13 values) of the methyl esters also favoured plants over animals as the lipid source. In the search for biomarkers by GC coupled to mass spectrometry on a silylated extract, a range of diacids together with high concentrations of 13,14-dihydroxydocosanoate and 11,12-dihydroxyeicosanoate were found. These compounds are oxidation products of erucic acid and gondoic acid, which are abundantly present in seeds of Brassicaceae plants. Liquid chromatography coupled to mass spectrometry analysis showed low concentrations of unaltered triglycerides, but revealed sizeable amounts of triglycerides with at least one dihydroxylated acyl chain. The unusual preservation of dihydroxylated triglycerides and alpha,omega-dicarboxylic acids can be related to the dry preservation conditions. Analysis of the stereoisomers of the dihydroxylated fatty acids allows one to determine whether oxidation took place during burning of the fuel or afterwards. The results prove that the oil of rapeseed (Brassica napus L.) or radish (Raphanus sativus L.) was used as illuminant in early Islamic Egypt, and that not only ceramic lamps but also mollusk shells were used as fuel containers. ispartof: Analytical and Bioanalytical Chemistry vol:390 issue:2 pages:783-793 ispartof: location:Germany status: published

The discovery that bile acids are involved in the modulation of nuclear steroid receptors has prompted renewed interest in this field of research. Due to the nature of research in this field, a technique that enables simple and effective assessment of the hydrophobic/hydrophilic balance, thus improving and speeding up evaluations of the biological profiles of these compounds, is greatly needed. In this context, both CMC value determination and RP-HPLC mobility evaluation were explored as possible approaches. While the CMC was calculated using the noninvasive conductimetric method, the RP-HPLC mobility was assessed by measuring the retention factor at several mobile phase compositions and extrapolating back to the pure aqueous mobile phase. The correlation of the CMC with the derived chromatographic hydrophobic index phi0 was satisfactory.