High-resolution Orbitrap Mass Research Articles

Identification of N-terminal protein processing sites by chemical labeling mass spectrometry.

Proteins undergo post-translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N-terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist. The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer. Sixteen examples of Staphylococcus aureus secretory proteins that lose an N-terminal signal peptide during their export were identified using this amidination approach. The N-termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered. A simple N-terminal amidination based mass spectrometry approach is described that facilitates identification of the N-terminus of a mature protein and the discovery of unexpected processing sites.

Characterization of metabolites of larotaxel in rat by liquid chromatography coupled with Q exactive high-resolution benchtop quadrupole orbitrap mass spectrometer

1. Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans for drug metabolism studies. Larotaxel is a taxane analog that has the potential for the treatment of various types of cancer.2. In this study, the metabolism of larotaxel was evaluated after an intravenous dose of 8 mg/kg via the caudal vein to healthy rats and its metabolites were characterized by high performance liquid chromatography coupled with a Q Exactive high-resolution benchtop quadrupole orbitrap mass spectrometer. Rat bio-samples were separated on a Capcell Pak C18 column (2.1 i.d. × 100 mm; 2.7 μm) with mobile phase of acetonitrile and water.3. As a result, a total of 34 metabolites were detected and identified by comparing the molecular masses, retention times and spectral patterns of the analytes with those of the parent drug. Three metabolites were confirmed by comparison with reference substances.4. The prominent metabolites were mainly hydroxyl, dihydroxyl, trihydroxyl and 10-desacetyl analogs of larotaxel, some of which resulted from oxidation of the tert-butyl groups on the side chain and further oxidation and cyclization of the tert-butyl hydroxylated metabolites.

Rapid screening for pesticides using automated online sample preparation with a high-resolution benchtop Orbitrap mass spectrometer

For pesticide analysis in food products a common approach is to develop a fast multi-residue method that is capable of identifying and quantifying a large number of analytes in various matrices. This study demonstrates rapid screening and accurate mass confirmation of 116 pesticides in oranges and hazelnuts using an automated online sample preparation method with turbulent-flow chromatography technology coupled to a high-resolution benchtop Orbitrap™ mass spectrometer. The limits of quantification (LOQs) for the majority of analytes are well below the maximum residue limit (MRL) set by the European Union and the Japanese government. The recoveries were in the range of 70–120% for over 75% of analytes in both matrices. The present methodology is suitable for routine pesticides analysis in food safety laboratories.