Chiara Guarino

Identification of changes in Triticum durum L. leaf proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum ‘Ofanto’). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4–7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed.

Development of an RP-HPLC method for the simultaneous determination of benzoic acid, sorbic acid, natamycin and lysozyme in hard and pasta filata cheeses.

A single method, based on RP-HPLC with UV detection, was developed with the aim of simultaneously quantifying four preservatives in cheeses: benzoic acid, sorbic acid, natamycin and lysozyme. The preservatives were extracted from different cheeses by using the same procedure, and separated by a single RP-HPLC gradient elution showing good resolution, in a short time. Recoveries were always higher than 91%; MDLs ranged from 0.4 to 4.0μgg(-1), and MQLs were included between 1.3 and 13.3μgg(-1); RDS ranged from 1% to 7%. Quantitation was performed in reference to a matrix matched calibration curve. The method was also applied to real samples for the determination of the four preservatives, with satisfying results.

Peptidomic approach, based on liquid chromatography/electrospray ionization tandem mass spectrometry, for detecting sheep's milk in goat's and cow's cheeses

A common fraud in the dairy field is the addition of sheeps milk to goats cheeses, because it has a very similar taste to goats milk, but is more available, and is commonly considered to have a better capacity to curdle. For similar reasons, and due to economic convenience, sheeps cheeses may also contain fraudulent cows milk. In order to detect this fraud, an EU official method may be used, but it is only a qualitative method (presence/absence of cows milk). A method able to quantify the presence of sheeps milk during cheese production in goats and cows cheeses was developed. The method is based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis of peptides of a casein extract from the cheese. By a simple procedure, caseins are extracted from cheeses, solubilized, digested with plasmin, and subsequently analyzed by LC/ESI-MS/MS. A typical sheeps peptide produced by plasmin hydrolysis (m/z = 860) was accurately selected and analyzed to understand if, and by how much, a declared pure goats cheese contains sheeps milk. By analyzing the same peptide it is also possible to detect if, and by how much, a declared pure sheeps milk contains, or not, cows milk. The method was applied to several goats and cows cheese samples. Quantitation was performed with a calibration curve obtained by analyzing curd cheeses containing different percentages of sheeps milk. The method detection limit and method quantitation limit were evaluated. This method appears accurate and suitable for detecting up to 2% of sheeps milk in cheeses.

Multi-detection of preservatives in cheeses by liquid chromatography–tandem mass spectrometry

The incorrect use of preservatives in cheeses may compromise food safety and damage consumers. According to the law, more than one preservative may be contemporarily used in cheeses. So a method for their contemporary detection may be useful for both manufacturers and control agencies quality control. In this research a liquid chromatography-tandem mass spectrometric with electrospray ionization method for the multi-determination of seven preservatives (benzoic acid, citric acid, hexamethylenetetramine, lysozyme, natamycin, nisin and sorbic acid) in cheese was developed. The preservatives were contemporarily extracted from cheese by a single procedure, and analyzed by RP-LC/ESI-MS/MS (Ion Trap) in positive ionization mode, with single reaction monitoring (SRM) acquisition. Three sample types (hard, pasta filata and fresh cheese) were used for method evaluation. Recoveries were mostly higher than 90%; MDLs ranged from 0.02 to 0.26 mgkg(-1), and MQLs were included between 0.07 and 0.88 mgkg(-1). Due to matrix effect, quantitation was performed by referring to a matrix matched calibration curve, for each cheese typology. This method was also applied to commercial cheese samples, with good results. It appears fast, reliable and suitable for both screening and confirmation of the presence and quantitation of the preservatives in a single, multi-detection analysis.

Absolute quantification of cardiac troponin T by means of liquid chromatography/triple quadrupole tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric method for absolute quantification of cardiac troponin T (cTnT) in mouse heart tissue is presented. Even in such a complex biological sample, the multiple reaction monitoring acquisition mode allowed the selective and sensitive determination of a specific peptide, obtained by cTnT enzymatic digestion. The concentration of this cTnT-specific peptide was considered as a representation of the concentration of its parent protein. Quantification was carried out by means of the matrix-matched calibration curve, constructed by adding the synthetic standard of the target peptide and another synthetic structurally analogous peptide as internal standard. Method identification limit and method quantification limit were estimated as 60 and 110 ng of cTnT per mg of total extracted proteins, respectively. The developed label-free approach has been applied for the absolute quantitation of cTnT because of its diagnostic and prognostic value as cardiac disease marker. However, the method could be of general application, since it requires only the synthesis of two suitable peptides, a protein tryptic cleavage product and an internal standard.