Cristiana Labella

Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI-FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in-source ion trap collision-induced dissociation (CID) of the protonated molecules, [M+H](+). A retro-Diels-Alder (RDA) process along with ring-contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH(3)NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO(2) or CO. The uracil derivatives showed a loss of a ketene unit (CH(2)CO, 42 Da) from the protonated molecule along with the loss of H(2)O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N-acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake.

Identification of two arginine kinase forms of endoparasitoid Leptomastix dactylopii venom by bottom up-sequence tag approach.

Leptomastix dactylopii (Howard) is an endoparasitoid wasp, natural enemy of mealybug Planococcus citri (Risso). Despite the acquired knowledge regarding this host-parasitoid interaction, only little information is available on the factors of parasitoid origin able to modulate the mealybug physiology. The major alteration observed in P. citri is a strong reduction in fecundity, which is evident soon after parasitization by L. dactylopii or venom injection in unparasitized hosts indicating that this proteinaceus secretion injected at the oviposition plays a key-role in host regulation. Protein identification of L. dactilopii venom has been limited by the lack of literature sources and public protein databases. Here, we identified two venom proteins by an integrated trascriptomic and proteomic approach. A custom-made transcriptomic database from the L. dactylopii venom glands was created by applying the high-throughput RNA sequencing approach. Two-dimensional gel electrophoresis (2DE) trypsinized protein spots were analyzed by high-resolution mass spectrometry (FTICRMS-12 T). The most abundant peptide ions were fragmented by collision induced dissociation and the obtained sequence tags were subjected to custom-made protein database searching. Two putative arginine kinases (full-length and truncated form) were identified. This is the first case in which both, truncated and full length arginine kinases, are identified in an endoparasitoid non-paralyzing venom.