Carlo G. Zambonin

Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin-based and HILIC-based affinity enrichment techniques, alone or in combination, for preparation of glycoproteins and glycopeptides for subsequent analysis by MALDI and ESI mass spectrometry. The methods were successfully applied to human serum samples and a total of 86 N-glycosylation sites in 45 proteins were identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer patients and healthy individuals to assess glycosylation site frequencies.

Determination of triazines in soil leachates by solid-phase microextraction coupled to gas chromatography–mass spectrometry

A solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed for the evaluation of the leachability order of selected triazines (propazine, terbuthylazine, sebuthylazine, ametryn, prometryn and terbutryn) in soil/sediment samples (organic carbon content ranging from 0.19 to 0.42%), analysing fractions collected from a soil packed microcolumn elution experiments. The procedure is fast, simple, highly sensitive and solvent free. SPME-GC-MS was also employed for the quantitative determination of triazines in the soil leachate, since the method showed good recovery yield. Detection limits were always better than 1 ng ml(-1). The method was tested on a contaminated landfill top soil. Prometryn and ametryn were identified through their MS spectra and then quantified. Terbuthylazine was used to assess recovery. Results compared well with those obtained by solvent extraction followed by HPLC-UV detection.

Solid-phase microextraction and gas chromatography–mass spectrometry for the rapid screening of triazole residues in wine and strawberries

A solid-phase microextraction gas chromatography-mass spectrometry method has been developed for the determination of triazole residues, such as triadimefon, propiconazole, myclobutanil and penconazole. The method has been successfully applied to the analysis of strawberries and wine samples. The procedure is solvent-free, simple and highly sensitive. Within-day and day-to-day RSDs ranged between 2-11% and 7-28%, respectively. Detection limits estimated at a signal-to-noise ratio of 3 ranged between 30 (propiconazole) and 100 ng/kg (triadimefon). Since the detection limits achieved by this method are well below the maximum residue levels for wine (or grapes) and strawberries recommended by the European legislation, it can be conveniently used as a low-cost rapid screening method for the contamination of the considered samples.

MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent

The adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HO) is frequent and constitutes a serious concern both for oil suppliers and consumers. The high degree of similarity between the two oils as regards triacylglycerol, total sterol and fatty acid profile, complicates the detection of low percentages of HO in EVOO. However, phospholipids (PLs) are usually present in seed oils at a concentration range of 10-20 g/kg, while the amounts of PLs in VOOs are 300-400 times lower. Thus, in this work a sample pretreatment procedure focused towards the selective PLs extraction was developed; the Bligh-Dyer extraction procedure was modified introducing the ionic liquid resulting from the combination of TBA (tributylamine) and CHCA (α-Cyano-4-hydroxycinnamic acid) as extraction solvent. The selective extraction and enrichment of phospholipids from EVOO and HO samples was then achieved. The relevant extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the same ionic liquid TBA-CHCA as MALDI matrix, that was found to be very suitable for PLs analysis. In fact, a remarkable increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, was observed in the relevant mass spectra. The applicability of the whole method to the individuation of the presence of HO in EVOO was demonstrated by the analysis of EVOO samples progressively adulterated with variable quantities of HO, that was still detectable at a 1% contamination level.

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.

Lactate Amperometric Biosensor Based on an Electrosynthesized Bilayer Film with Covalently Immobilized Enzyme

An alternative approach to the fabrication of an amperometric biosensor, which combines electrochemical polymerization of a bilayer film and covalent binding of the enzyme, is presented. A Pt electrode modified by an anti-interferent poly(pyrrole) layer is covered by a poly(tyramine) film, exposing amino groups that are then used for the covalent attachment of lactate oxidase after activation with glutaraldehyde. In this way, a fabrication procedure is obtained that combines the advantages of electrochemical polymerization (high spatial control, no restrictions in electrode shape and dimension, reproducible film thickness even in the sub-micrometre range) with those of covalent binding of the enzyme (no enzyme loss, higher response stability and increased lifetime of the sensor). The interferent rejection efficiency is dictated by the diffusional barrier provided by the bilayer membrane and by the degree of permselectivity that can be built up in the poly(pyrrole) film. A peculiar property was displayed by the bilayer, i.e., the ability to separate L-lactate and residual interferent responses in the time domain in both flow injection and batch addition experiments. The resulting L-lactate amperometric biosensor was characterized in terms of sensitivity, response time, linear response range, stability and rejection of electroactive interferents.

Solid-phase microextraction-gas chromatography mass spectrometry and multivariate analysis for the characterization of roasted coffees.

HS-SPME-GC-MS in combination with PCA was employed to discriminate different arabica/robusta blends having different geographical origins. HS-SPME confirmed to be an effective and reproducible sampling technique for routine characterization of coffees. In addition, the chemometric approach permitted to find parameters suitable for the differentiation of the different blends and the determination of the real quality of the products. Finally, the proposed methods have been successfully applied to some commercial coffee blends.

Determination of sugars and alditols in food samples by HPAEC with integrated pulsed amperometric detection using alkaline eluents containing barium or strontium ions

Abstract The addition, in mobile phase, of alkaline-earth divalent cations such as Sr(II) and Ba(II) is shown to be a versatile means of improving the separation of alditols and carbohydrates, which are conventionally determined by high-performance anion-exchange chromatography (HPAEC) coupled with integrated pulsed amperometry using gold working electrodes. It is believed that Ba(II) or Sr(II) ions are effective both in forming solution complexes with sugar analytes, and ensuring thoroughly carbonate-free alkaline mobile phase flowing through the column. As a result, substantial benefits can be obtained in terms of peak shape, attainable column efficiency, and detection sensitivity. This work describes determination of alditols (myo-inositol, d -sorbitol, and d -mannitol), and carbohydrates (glucose, fructose, and sucrose) in some common fruits and fruit products (apricot, plum, watermelon, lemon, mandarin, grape, and dietetic cherry jam) and vegetable foodstuffs (cauliflower, cucumber, fennel, tomato, turnip, and celery). The free sugar contents in fruit juices were determined directly, without pretreatment or derivatization, following a sample extraction. While myo-inositol was found in many fruits, fruit products and vegetables examined, no presence of xylitol was detected.

Determination of Ochratoxin A in green coffee beans by solid-phase microextraction and liquid chromatography with fluorescence detection

A new method for the determination of Ochratoxin A (OTA) in green coffee beans by solid-phase microextraction (SPME) coupled to liquid chromatography with fluorescence detection (LC-FD) is described for the first time. Coffee samples were extracted by a 5% NaHCO(3) solution, followed by a clean-up step of the extract by chloroform partition. The aqueous extract was then acidified and finally subjected to SPME-LC-FD analysis. The investigated linear range in coffee was 2-32 ng/g. Within-day RSD% in coffee spiked at 2 and 32 ng/g levels were 3.3 and 2.7, respectively, whereas the between-days RSD% were 4.1 and 3.8, respectively. The limits of detection (LOD) and quantitation (LOQ), calculated at a signal-to-noise ratio of 3 and 10 (noise calculated peak to peak on a blank chromatogram at the OTA retention time), were 0.3 and 2 ng/g, respectively.

Proteomic Approach Based on MALDI-TOF MS To Detect Powdered Milk in Fresh Cow’s Milk

Milk and cheese are expensive foodstuffs, and their consumption is spread among the population because of their high nutritional value; for this reason they are often subjected to adulterations. Among the common illegal practices, the addition of powdered derivatives seems very difficult to detect because the adulterant materials have almost the same chemical composition of liquid milk. However, the high temperatures (180-200 °C) used for milk powder production could imply the occurrence of some protein modifications (e.g., glycation, lactosylation, oxidation, deamidation, dehydration). The modified proteins or peptides could then be used as markers for the presence of powdered milk. In this work, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze tryptic digests relevant to samples of raw liquid (without heat treatment), commercial liquid, and powdered cows milk. Samples were subjected to two-dimensional gel electrophoresis (2-DE); differences among liquid and powder milk were detected at this stage and eventually confirmed by MALDI analysis of the in gel digested proteins. Some diagnostic peptides of powdered milk, attributed to modified whey proteins and/or caseins, were identified. Then, a faster procedure was optimized, consisting of the separation of caseins from milk whey and the subsequent in-solution digestion of the two fractions, with the advantage of obtaining almost the same information in a limited amount of time. Finally, analyses were carried out with the fast procedure on liquid milk samples adulterated with powdered milk at different percentages, and diagnostic peptides were detected down to 1% of adulteration level.