Benito Cañas

Species Differentiation of Seafood Spoilage and Pathogenic Gram-Negative Bacteria by MALDI-TOF Mass Fingerprinting

Species differentiation is important for the early detection and identification of pathogenic and food-spoilage microorganisms that may be present in fish and seafood products. The main 26 species of seafood spoilage and pathogenic Gram-negative bacteria, including Aeromonas hydrophila, Acinetobacter baumanii, Pseudomonas spp., and Enterobacter spp. among others, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of low molecular weight proteins extracted from intact bacterial cells by a fast procedure. From the acquired spectra, a library of specific mass spectral fingerprints was constructed. To analyze spectral fingerprints, peaks in the mass range of 2000-10 000 Da were considered and representative mass lists of 10-35 peak masses were compiled. At least one unique biomarker peak was observed for each species, and various genus-specific peaks were detected for genera Proteus, Providencia, Pseudomonas, Serratia, Shewanella, and Vibrio. Phyloproteomic relationships based on these data were compared to phylogenetic analysis based on the 16S rRNA gene, and a similar clustering was found. The method was also successfully applied for the identification of three bacterial strains isolated from seafood by comparing the spectral fingerprints with the created library of reference fingerprints. Thus, the proteomic approach demonstrated to be a competent tool for species identification.

Elemental Bioimaging in Kidney by LA–ICP–MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

Ym1 Is a Neutrophil Granule Protein That Crystallizes in p47 phox -deficient Mice

Crystals were discovered within the aged lung and at sites of chronic inflammation in a mouse model of chronic granulomatous disease. Following re-crystallization at neutral pH, the crystals were identified as the chitinase-like protein Ym1, expressed in organs of the lymphoreticular system, the lung, and distal stomach. Ym1 was shown to be a neutrophil granule protein and to have weak β-N-acetylglucosaminidase activity, indicating that it might contribute to the digestion of glycosaminoglycans. Crystal formation is likely to be a function of excess neutrophil turnover at sites of inflammation in the chronic granulomatous disease mouse. Failure to remove subcutaneous Ym1 crystals injected into knockout mice indicates that a failure of digestion may also contribute to crystallization.

Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry

A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.

Human proteome enhancement: High-recovery method and improved two-dimensional map of colostral fat globule membrane proteins

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2 – 4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two‐dimensional electrophoresis (2‐DE) map of the human milk fat globule membrane proteins, both integral and membrane‐associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3 – 10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on 11.5% T homogeneous gels. A reproducible 2‐DE map of integral and membrane‐associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis and/or by amino acid sequencing.

Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting

The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram‐positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10–30 peak masses. Genus‐specific as well as species‐specific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web‐application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI‐TOF MS fingerprinting is applied to Gram‐positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.

Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils

Neutrophils play a key role in host-defence mechanisms against invading pathogens, using their capacity to migrate, engulf micro-organisms and produce toxic radicals. Protein kinase C (PKC) isotypes are important intracellular regulators of these processes in neutrophils. PKC isotypes themselves are controlled by interactions with lipids, Ca(2+) and proteins. The C2-like domain of PKC-delta (deltaC2) has been identified as a protein-interaction domain in this PKC isotype. In the present paper we have investigated the contribution of protein interactions at this domain to the regulation/function of PKC-delta in neutrophils. Using affinity chromatography we identified actin as a deltaC2 binding partner in these cells. Fluorescein-labelled deltaC2, microinjected into immobilized neutrophils, interacts with filamentous actin (F-actin) inside the cell. PKC-delta co-localizes with F-actin in neutrophils, in lamellipodia at the leading edge of the cell. Stimulation with phorbol ester or IgG-opsonized Staphylococcus aureus results in co-ordinated redistribution of PKC-delta and F-actin, and a PKC-delta inhibitor inhibits these changes. Microinjection of deltaC2 also inhibits F-actin redistribution. Thus PKC-delta binds to F-actin through its C2 domain, and these interactions are important in regulating actin redistribution in neutrophils.

Mass Spectrometry Characterization of Species-Specific Peptides from Arginine Kinase for the Identification of Commercially Relevant Shrimp Species

The identification of commercial shrimp species is a relevant issue to ensure correct labeling, maintain consumer confidence and enhance the knowledge of the captured species, benefiting both, fisheries and manufacturers. A proteomic approach, based on 2DE, tryptic in-gel digestion, MALDI-TOF MS, and ESI-MS/MS analyses, is proposed for the identification of shrimp species with commercial interest. MALDI-TOF peptide mass fingerprint from arginine kinase tryptic digests were used for the identification of seven commercial, closely related species of Decapoda shrimps. Further identification and characterization of these peptides was performed by CID on an ESI-IT instrument, database search and de novo sequence interpretation, paying special attention to differential, species-specific peptides. Fisheries and manufacturers may take advantage of this methodology as a tool for a rapid and effective seafood product identification and authentication, providing and guaranteeing the quality and safety of the foodstuffs to consumers.

Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring.

A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers.

Rapid direct detection of the major fish allergen, parvalbumin, by selected MS/MS ion monitoring mass spectrometry.

Parvalbumins beta (β-PRVBs) are considered the major fish allergens. A new strategy for the rapid and direct detection of these allergens in any foodstuff is presented in this work. The proposed methodology is based on the purification of β-PRVBs by treatment with heat, the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of only nineteen β-PRVB peptide biomarkers by Selected MS/MS Ion Monitoring (SMIM) in a linear ion trap (LIT) mass spectrometer. The present strategy allows the direct detection of the presence of fish β-PRVBs in any food product in less than 2 hours.