N-Glycomics of Human Erythrocytes.

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.

Polycyclic aromatic hydrocarbons in Mullus surmuletus from the Catania Gulf (Sicily, Italy): distribution and potential health risks.

Different specimens of Mullus surmuletus from the Catania Gulf (Sicily) were sampled and analysed for the quantification of 16 priority polycyclic aromatic hydrocarbons (PAHs) in order to evaluate the distribution of these pollutants and the potential human health risks associated to the consumption of fish. The determined PAHs concentration values ranged from 0.25 to 6.10 ng/g wet weight and the most abundant PAHs detected were lower molecular weight (LMW) compounds with 2 to 3 rings. Relying on the PAHs concentration values, on the consumption data and on the total toxic equivalent (TEQ), the incremental lifetime cancer risk (ILCR) was assessed and its calculated value (2.97 × 10-7) is far below the "maximum acceptable risk level" (ARL), suggesting a low potential carcinogenic risk on consuming M. surmuletus for local population. Even though the study shows a quite low contamination level in M. surmuletus, intensive monitoring programmes are still highly needed in order to provide a better picture of the PAHs distribution in Catania Gulf and of the human health risk linked to fish consumption.

CSF N-Glycoproteomics Using MALDI MS Techniques in Neurodegenerative Diseases.

CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases. To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer's disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.

CSF N-Glycomics Using MALDI MS Techniques in Alzheimer's Disease.

In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease.

CSF N-glycoproteomics for early diagnosis in Alzheimer's disease.

This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer's disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes.

Determination by LC-MS/MS of colistins A and B in plasma and ultrafiltrate from critically ill patients undergoing continuous venovenous hemodiafiltration.

BACKGROUND: Colistin is a 50-year-old antibiotic, the use of which was ceased in the 70s and recently resumed as a "salvage therapy" against multidrug-resistant gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii. The narrow therapeutic range of colistin makes the choice of its correct dosage crucial, and monitoring of blood concentration is occasionally necessary for critically ill patients, including intensive care patients subjected to continuous renal replacement therapy. METHODS: Two LC-MS/MS methods were developed and fully validated for the quantitative determination of colistins A and B in plasma and dialysis ultrafiltrate (UF) samples, ultimately arising from 4 patients undergoing continuous venovenous hemodiafiltration (CVVHDF). RESULTS: The developed methods proved to be both specific and selective. They showed good fit and linearity over the entire range of interest. Trueness and accuracy proved satisfactory. Both methods have excellent intraassay precision (percent coefficient of variations were lower than 10%) and limit of detection values in the range 20-100 ng/mL, about 1-2 orders of magnitude below the concentrations commonly detected in real samples. The mean sieving coefficient (SC) values, measured after 10 minutes of CVVHDF, were 0.42 for colistin A and 0.48 for colistin B. SC values proved to be quite stable for 24 hours, but then declined to 0.24 for colistin A and 0.32 for colistin B, respectively, after 48 hours. At the median blood flow and effluent flow rate of 120 and 28 mL/min, clearance values for colistin B were higher than 15 mL/min. During the entire duration of CVVHDF sessions, the SC and clearance values for colistin A were significantly lower than colistin B. CONCLUSIONS: Two simple methods for the simultaneous determination of colistins A and B have been developed and validated. Their application in the clinical setting demonstrates that CVVHDF treatment lasting 48 hours produces a relatively constant and efficient removal of the drug.