A carbonyl-reactive photo-cleavable isobaric labeling affinity tag can provide a selective, high-throughput, and reproducible approach for the quantitative analysis of aldehyde and ketone metabolites in complex biological samples.

Carbapenems are broad spectrum antibiotics considered as a “last resort” medicine to treat bacterial infections. Carbapenem-hydrolyzing β-lactamases (also called carbapenemases), however, can confer bacterial resistance and represent a serious health threat. Here, we report a novel approach using 18O labeling and selected reaction monitoring to detect carbapenemase activity from pathogenic microorganisms in a rapid and quantitative manner. Four model bacterial strains bearing various classes of β-lactamases were tested for their capability to hydrolyze Meropenem, an FDA-approved carbapenem drug. We were able to predict the Meropenem resistance of these bacteria on the basis of their carbapenemase activity, suggesting the great potential of our method in clinical diagnostics.

Isobaric tags have broad applications in both basic and translational research, as demonstrated by the widely used isobaric tag for relative and absolute quantitation (iTRAQ). Recent results from large-scale quantitative proteomics projects, however, indicate that protein quantification by iTRAQ is often biased in complex biological samples. Here, we report the application of another isobaric tag, deuterium isobaric amine reactive tag (DiART), for quantifying the proteome of Thermoanaerobacter tengcongensis (T. tengcongensis), a thermophilic bacterium first discovered in China. We compared the performance of DiART with iTRAQ from several different aspects, including their fragmentation mechanisms, the number of identified proteins, and the accuracy of quantification. Our results revealed that, as compared with iTRAQ, DiART yielded significantly stronger reporter ions, which did not reduce the number of identifiable peptides, but improved the signal-to-noise ratio (S/N) for quantification. Remarkably, we found that, under identical chromatography and mass spectrometry (MS) conditions, DiART exhibited less reporter ions ratio compression than iTRAQ, probably due to more reporter ions with higher intensities produced by DiART labeling. Taken together, we demonstrate that DiART is a valuable alternative of iTRAQ with enhanced performance for quantitative proteomics.

Protein–fragment complementation assay using a hydrolysis-deficient β-lactamase provides a robust tool for studying protein–protein interactions.

This paper demonstrates the applications of a novel isobaric reagent, named deuterium (2H) isobaric amine-reactive tag (DiART), for quantitative proteomics. Peptides labeled with DiART were analyzed using an electrospray ionization (ESI)-based LTQ-Orbitrap mass spectrometer. Our data showed that 2H-associated isotope effects, such as partial loss of 2H labels during tandem mass spectrometry (MS/MS) and 2H-related chromatographic shift, were either not observed or negligible. With the use of a hybrid collision-induced dissociation (CID)−higher energy C-trap dissociation (HCD) acquisition method, we were able to identify DiART-labeled peptides with high confidence and quantify them with high accuracy. Furthermore, we adopted a hybrid electron-transfer dissociation (ETD)−HCD acquisition protocol and developed a novel data analysis approach to measure phosphorylation of peptides. Our results showed DiART had excellent performance on LTQ-Orbitrap instruments and provided a cost-effective technique for large-scale quantitative proteomics measurements.

Protein–fragment complementation assay using a hydrolysis-deficient β-lactamase provides a robust tool for studying protein–protein interactions.

A carbonyl-reactive photo-cleavable isobaric labeling affinity tag can provide a selective, high-throughput, and reproducible approach for the quantitative analysis of aldehyde and ketone metabolites in complex biological samples.