Anna Natale

Strategies in protein sequencing and characterization: multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry.

A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.

Development of an analytical method for the determination of polyphenolic compounds in vegetable origin samples by liquid chromatography and pulsed amperometric detection at a glassy carbon electrode

A sensitive and accurate method for the determination of polyphenolic compounds in artichoke bract extracts and olive mill wastewaters by liquid chromatography coupled with pulsed amperometric detection at a glassy carbon working electrode was developed. Preliminary experiments were carried out by cyclic voltammetry to investigate the electrochemical behavior of polyphenols under different mobile phase compositions, and to test the detection and cleaning electrode potentials. Chromatographic separations were performed by using a core-shell C18 column, eluted with acetic acid and acetonitrile, by combined concave-linear binary gradients. Under the optimized experimental conditions, a good column efficiency and peak symmetry were observed, also for stereo and positional isomeric compounds. The developed three-step potential waveform for pulsed amperometric detection was successfully applied for the sensitive chromatographic determination of polyphenols in artichoke extracts and olive mill wastewaters. Linearity, precision and sensitivity of the proposed method have been evaluated. A wide linear range of response (up to 20 mg/L) has been obtained for all the investigated compounds. Detection and quantification limits in the vegetable origin sample extracts were in the range 0.004-0.6 mg/L and 0.01-2mg/L, respectively, while the injection-to-injection repeatability (n=6) ranged from 5 to 13%. The obtained results confirmed the excellent sensitivity of the electrochemical detection, and its suitability for the determination of electroactive polyphenolic compounds at low concentration levels.

Pulsed amperometric detection at glassy carbon electrodes: A new waveform for sensitive and reproducible determination of electroactive compounds

In this work, the application of a new pulsed amperometric detection (PAD) waveform at a glassy carbon electrode, operating in typical chromatographic mobile phases, is proposed for the sensitive and reproducible determination of arylethanolaminic and phenolic moiety based compounds (e.g. beta-agonists and polyphenols). Preliminary experiments by cyclic voltammetry were carried out to investigate the electrochemical behaviour and to select the detection and cleaning electrode potentials. The proposed potential-time profile was designed to prevent the carbon electrode fouling under repeated analyses, thus ensuring a reproducible and sensitive quantitative determination, without the need of any mechanical or chemical electrode cleaning procedure. The waveform electrochemical parameters, including detection and delay times, were optimized in terms of sensitivity, limit of detection and response stability. The optimized waveform allowed the sensitive and stable detection of model compounds, such as clenbuterol and caffeic acid, that showed detection limits of 0.1 μg L(-1) and 14 μg L(-1), quantification limits of 0.4 μg L(-1) and 46 μg L(-1), and linearity up to 100 μg L(-1) (r = 0.9993) and 10 mg L(-1) (r = 0.9998), respectively. Similar results were obtained for other compounds of the same classes, with precision values under repeatability conditions ranging from 3.0 to 5.9%. The proposed method can be then considered as an excellent alternative to the post-column detection of beta-agonists, phenols and polyphenols.

Milk authenticity by ion-trap proteomics following multi-enzyme digestion

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity. In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine β-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity.

Combined use of peptide ion and normalized delta scores to evaluate milk authenticity by ion-trap based proteomics coupled with error tolerant searching

A fundamental issue in proteomics is the peptide identification by database searching and the assessment of the goodness of fit between experimental and theoretical data. Despite the different number of ways to measure the quality of search results, the definition of a scoring criterion is still highly desirable in ion-trap based proteomics. Indeed, in order to fully take advantage of a low resolution MS/MS dataset, it is essential to strike a balance between greater information capture and reduced number of incorrect peptide assignments. In addition, the development of user-specified rules is a crucial aspect when very similar proteins of the same family are analyzed in order to infer the origin species. In this study, a post-processing validation scheme is provided for the evaluation of proteomic data in shot-gun ion-trap proteomics, when a flexible database searching based on the error tolerant mode is adopted in combination with a low-specificity enzyme to maximize sequence coverage. To validate peptide assignments, we used standard β-casein digested with trypsin/chymotrypsin or trypsin alone and the popular search engine MASCOT to identify the relevant (known) peptide sequences. A linear combination between peptide ion score and normalized delta score (i.e. the difference between the best and the second best ion score, divided by the best score) is proposed to increase the accuracy in sequence assignments from low-resolution tandem mass spectra. Finally, the optimized post-processing database validation was successfully applied to the direct analysis of milk tryptic/chymotryptic digests of different origin, without resorting to two-dimensional electrophoresis that is usually performed for protein separation in ion-trap proteomics. The identification of species-specific amino acidic sequences among the validated peptide spectrum matches has allowed to fully discriminate between the animal species, so evaluating accurately the milk authenticity.

Characterization of Silter Cheeses Produced in Valley and Alpine Pastures by a Proteomic Approach

Silter is an Italian hard cheese manufactured with milk produced by cows fed at different altitude, valley or alpine pasture. The chemical, rheological and sensory properties of cheeses can be affected by the modification in milk composition due to the breed which at different altitude causes the modification of protein content, κ-CN glycosylation, plasmin activity, and coagulation properties. The influence of milk plasmin activity on dairy production was investigated in seven Silter cheeses, four produced in the valley and three from alpine mountain through alkaline urea-polyacrylamide gel electrophoresis; two-dimensional gel electrophoresis coupled to mass spectrometry and image analysis. Results demonstrated that Silter cheese obtained from cows reared in alpine pasture is characterized by a more evident proteolysis, determining high levels of β-CN and αs1-CN fragments. Therefore, the most relevant fragmentation was attributed to a more intense activity of plasmin and to a different dosage of rennet to make up for the reduced coagulation properties of alpine milk.

Mass spectrometry hyphenated techniques for the analysis of volatiles and peptides in soft cheese: Useful tools for the shelf life optimization.

In order to assess the product quality and shelf life of an Italian soft cream cheese under different storage conditions, the volatile and peptide profiles evolution were tested. Volatiles were sampled directly from the head space of cheese packaging by solid‐phase microextraction and analyzed by GC‐MS. Peptide profiles were obtained by nanoLC‐MS/MS, following a novel bioinformatics approach based on scoring distribution associated to the protein hits originating from the database search. In particular, a refined identification by focusing on selected time segments corresponding to the most intense peaks was carried out. A total of 40 compounds including acids, aldehydes, ketones, lactones, alcohols, esters, hydrocarbons, terpene, sulfur, and aromatic compounds were detected. Significant differences in their abundance during the storage in different packagings were observed, as well as an evolution of peptides mainly belonging to αS1‐casein. The results demonstrated the usefulness of the above‐mentioned hyphenated techniques for the determination of the soft cheese shelf life under different storage conditions.