The time course of the thylakoid membranes proteomic profile changes upon cadmium (Cd) addition to hydroponic Spinacia oleracea L. plants has been investigated. Two different proteomic approaches have been used: blue native gel electrophoresis followed by SDS-PAGE (2D BN-SDS-PAGE) and sucrose density gradient ultracentrifugation followed by RP-HPLC. Chlorophyll (Chl) and xanthophylls concentrations, together with ESR and real time PCR measurements, were also performed to get a complete overview of all photosystem changes. Cd only accumulated in basal leaves, that therefore were prevalently investigated for assessment of Cd induced changes. Here, Cd strongly reduced Chl concentration, especially Chl a. During the first 15 days of treatment, native electrophoresis system revealed high sensitivity of PSI to Cd, while minor effects on PSII were observed. Cytochrome b(6)/f and the ATP-synthase complex did not change following the Cd treatment. A significant reduction of antenna proteins of PSI was observed, while PSII antennae were affected to a minor extent, with exception of the isomeric Lhcb1.1 which decreased significantly already at the onset of the treatment. Some PSII core proteins were overexpressed, but showed reduced activity. No new protein was formed and no specific protein disappeared in the photosynthetic apparatus of Cd-treated leaves. Upon removal of Cd, a rapid resynthesis of total Chl and a significant resynthesis of Lhcb1.1 antenna were observed, suggesting that Cd affects specifically the photosynthetic apparatus of spinach basal leaves, replacing other metal ions inside proteins.

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A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.

dWe evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae. Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of bla KPCproducing K. pneumoniae showed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform. In the last decade, an emerging spread of carbapenemase-producing Enterobacteriaceae (CPE) was observed worldwide, especially in Klebsiella pneumoniae strains. As a result, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become endemic in several countries, including Italy, Greece, and the United States (1).Rapid detection of KPC producers is mandatory to limit the spread of this pathogen and for clinical management of complicated infection due to KPC-producing K. pneumoniae. Numerous phenotypic (e.g., Carba NP, modified Hodge test, disk-diffusion synergy test) and genotypic (e.g., single and multiplex PCR) assays were developed for rapid identification of CPE (2, 3). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was proposed to detect carbapenemase production in Gram-negative organisms (4).Here, we propose a simple approach based on MALDI Biotyper RTC software to identify KPC-producing K. pneumoniae clinical strains. The aim of this study was to evaluate the sensitivity and specificity of an innovative MALDI-TOF approach using a large population of well-characterized unrelated K. pneumoniae strains collected in Italy.␤-Lactamase genes and the clonal relatedness of bacterial isolates were investigated by PCR and multilocus sequence typing (MLST) analysis (3, 5). For MALDI-TOF MS analysis, strains were evaluated as previously described (6).Dendrogram analysis revealed two main separate clusters, representing KPC-producing and non-KPC-producing K. pneumoniae strains, which included extended-spectrum ␤-lactamase (ESBL) producers and wild-type strains (Fig. 1). In order to evaluate the major significant differences between the protein patterns of these two groups, spectra were analyzed by ClinProtTools software (v3.0; Bruker Daltonics, Inc.). Single-peak analysis identified a total of 10 peaks (m/z of 5. 555, 6.082, 6.152, 7.384, 7.663, 7.704, 7.763, 7.851, 11.109, and 11.185) that potentially differentiate between KPC-positive and KPC-negative K. pneumoniae strains. In order to identify group-specific peaks as candidate biomarkers for KPC-producing K. pneumoniae strains, the presence of different peaks were manually investigated by ClinProtTools software. Our analysis demonstrated that an 11.109-Da peak was detected in most of the spectra obtained from KPC-positive strains, whereas it was absent in all of the KPC-negative isolates. At the same time, the remaining eight peaks (excluding the 5.555-Da peak as a double charged 11.109-Da peak) were found...