Donatella Aiello

Review: multistage mass spectrometry in quality, safety and origin of foods.

Quality and safety control and the validation of origin are hot issues in the production of food and its distribution, and are of primary concern to food and agriculture organization. Modern mass spectrometry (MS) provides unique, reliable and affordable methodologies to approach with a high degree of scientificity any problem which may be posed in this field. In this review the contribution of mass spectrometry to food analysis is presented aiming at providing clues on the fundamental role of the basic principles of gas-phase ion chemistry in applied research fields. Applications in proteomics, allergonomics, glycomics, metabolomics, lipidomics, food safety and traceability have been surveyed. The high level of specificity and sensitivity of the MS approach allows the characterization of food components and contaminants present at ultra-trace levels, providing a distinctive and safe validation of the products.

Vegetable proteomics: the detection of Ole e 1 isoallergens by peptide matching of MALDI MS/MS spectra of underivatized and dansylated glycopeptides.

Ole e 1 (NCBI entry gi|14424429) is the major allergen of Oleaceae family. Multiple isoforms and variants are present in varying degrees of distribution. In this report, we present a new approach to the resolution of multiple forms of Ole e 1 from whole antigen extracts, based on a preliminary chemical fractionation procedure followed by MALDI MS and MS/MS measurements. The characterization of Ole e 1 isoallergens was accomplished through the identification of the amino acid sequence including the glycosylation site and the structure of the glycan moieties. The structure feature of the identified Ole e 1.0102 (gi|2465127), main olive allergen [Olea europaea] (gi|13195753), Major pollen allergen Ole e 1 (gi|33329740) and Ole e 1c (gi|1362131) is represented by the point mutation K(106) --> I and by the presence of a glycan moiety. Two other variants Major pollen allergen (Allergen Ole e1) (Ole e I) (gi|14424429) and Ole e 1.0103 protein [Olea europea] (gi|2465129) were identified as nonglycosylated species. These results, partially in disagreement with Swiss-Prot annotation, were validated by matching the MALDI MS/MS spectra of the natural tryptic mixture with those obtained after deglycosylation.

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry.

We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

Mass Spectrometry-Based Proteomic Approach in Oenococcus oeni Enological Starter

A simple procedure is proposed for selective protein solubilization and trypsin digestion, followed by off-line liquid chromatography-matrix assisted laser desorption ionization mass spectrometry (LC-MALDI MS) analysis of Oenococcus oeni (O. oeni) bacterium. Peptides were identified from tryptic digests using sequencing by tandem mass spectrometry and database searches. Cytoplasmic and membrane related proteins (MRP) were identified in the O. oeni bacterium. MS/MS data analysis points out 13 peptides having one point mutation from 9 proteins. The major microheterogeneity was found for Zn-dependent alcohol dehydrogenase (Zn-ADH, Q04GE6) and 60 kDa chaperonin (GroEL, Q04E64) that are involved in methionine catabolism and post-translational protein folding, respectively. MS/MS data processing also leads to the identification of 34 unique phosphorylation sites from 19 phosphoproteins.

Solid Phase Isobaric Mass Tag Reagent for Simultaneous Protein Identification and Assay

The solid phase isobaric mass tagging (SPIMT) approach is presented for simultaneous protein quantitation and identification. The novelty of the SPIMT strategy relies on a CID-based differentiation of regioisomeric species for quantitation of tagged proteolytic peptides. SPIMTs are unlabeled mass-tagging reagents, which consist of a reporter group, a mass balance group, and a spacer with a amine-specific reactive group, able to be linked to any N-terminal peptide. Therefore SPIMT-linked peptides from a two-plex set appear as a single unresolved precursor ion in MS, whereas the reporter groups lead to quantitation signals of m/z 168.2 and 182.2 Da upon tandem mass spectrometry (MS/MS) analysis with matrix-assisted laser desorption time-of-flight/time-of-flight (MALDI TOF/TOF). This strategy allows ease protein identification by direct submission of MS and MS/MS data to the MASCOT database. SPIMT approach showed an excellent quantitation linearity, detecting any relative concentration differences of peptides in two solutions over a 5-fold concentration range without losing sequencing information. Therefore, SPIMTs are an attractive, simple, and low cost alternative for two-plex quantitation of proteins and offer possibilities of tuning the two-plex signal mass window by replacing the spacer.

N‐hydroxysuccinimidyl p‐methoxybenzoate as suitable derivative reagent for isotopic dilution assay of biogenic amines in food

We present a simple methodology for the simultaneous identification and determination of biogenic amines in food matrices, based on the use of a stable isotope-coded derivatization and liquid chromatography tandem mass spectrometry. The tagging reagent is N-hydroxysuccinimidyl ester of d(0)/d(4) -4-methoxybenzoic acid (d(0)/d(4) -4-MBA-OSu) which mainly functionalizes primary amines. The identification and structural characterization of tagged biogenic amines were exploited by matrix-assisted laser desorption/ionization-mass spectrometry (MS) and MS/MS. Multiple-reaction monitoring has been applied in the assay of biogenic amines in different foodstuffs, providing a method whose reliability is confirmed by the values of accuracy (12%) and by the calculated analytical parameters.

Targeted proteomic approach in prostatic tissue: a panel of potential biomarkers for cancer detection

Prostate cancer (PCa) is the sixth highest causes of cancer-related deaths in men. The molecular events underlying its behavior and evolution are not completely understood. Prostate-specific antigen (PSA) is the only approved Food and Drug Administration biomarker. A panel of ten stage-specific tumoral and adjacent non tumoral tissues from patients affected by PCa (Gleason score 6, 3+3; PSA 10 ÷19 ng/ml) was investigated by MS-based proteomics approach. The proposed method was based on identifying the base-soluble proteins from tissue, established an efficient study, which lead to a deeper molecular perspective understanding of the PCa. A total of 164 proteins were found and 132 of these were evaluated differentially expressed in tumoral tissues. The Ingenuity Pathway Analysis (IPA) showed that among all dataset obtained, 105 molecules were involved in epithelial neoplasia with a p-value of 3.62E-05, whereas, only 11 molecules detected were ascribed to sentinel tissue and bodily fluids.

Human coelomic fluid investigation: A MS-based analytical approach to prenatal screening

Coelomic fluid (CF) is the earliest dynamic and complex fluid of the gestational sac. CF contains maternal cells and proteins produced by embryonic cells, tissues and excretions. The biochemical composition of CF is modified throughout the first trimester of pregnancy and its protein profile reflects both physiological/pathological changes affecting the embryo and mother. Identification of variations in the balance of proteins might indicate particular types of pathologies, or ascertain specific genetic disorders. A platform utilizing protein enrichment procedures coupled with shotgun identification and iTRAQ differentiation provided the identification and quantitation of 88 unique embryonic proteins. It is relevant to note that chromosome X protein CXorf23 was found suggesting the embryo sex. Foetal sex was determined by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) on coelomic cells, foetal tissues and maternal white blood cells, with a 100% concordance rate between iTRAQ-MS/MS and QF-PCR data. The functional associations among the identified proteins were investigated using STRING database. Open Targets Platform showed as significant the following therapeutic areas: nervous, respiratory, eye and head system disease.

Molecular species fingerprinting and quantitative analysis of saffron (Crocus sativus L.) for quality control by MALDI mass spectrometry

Herein we describe a rapid, simple, and reliable method for the quantitative analysis and molecular species fingerprinting of saffron (Crocus sativus L.) by direct MS and MS/MS analysis. Experimentally, powdered saffron was subjected to a brief treatment with a 0.3% TFA water/acetonitrile solution, and the resulting mixture was directly placed on the MALDI plate for analysis. This approach allowed the detection of the commonly observed crocins C-1–C-6 and flavonols, together with the identification of the unknown highly glycosylated crocins C-7, C-8 and C-9, and carotenoid-derived metabolites. The strategy endorsed the simultaneous detection and characterization of saffron and adulterant markers using crude extracts of the adulterant itself and synthetic sets of adulterated authentic saffron samples. The implementation of the strategy was to measure the amount of an unknown adulterant from the crude extract using curcumin as a non-isotopic isobaric internal standard. The relationship between the saffron and curcumin molar ratios were established with a correlation coefficient of 0.9942. The ANOVA regression model was significant, F(1, 72) = 13 595.82, p < 0.001, y = (0.0116 ± 0.0001)x + (−0.1214 ± 0.0086). No matrix effects were observed and good results were obtained with respect to instrumental repeatability (*RSD% < 2%) and LOD (1.1%). The analysis of commercial samples of saffron using the proposed approach showed the suitability of the method for routine analysis (minimal sample preparation and very short measuring time per sample).

Chemoselective Protection of Glutathione in the Preparation of Bioconjugates: The Case of Trypanothione Disulfide

A novel synthetic route to the chemoselectively protected N,S-ditritylglutathione monomethyl ester is described involving the chemical modification of the commercially available glutathione (GSH). The synthetic value of this building block in the facile preparation of GSH bioconjugates in a satisfying overall yield was exemplified by the case of trypanothione disulfide (TS2), a GSH-spermidine bioconjugate, involved in the antioxidative stress protection system of parasitic protozoa, such as trypanosoma and leishmania parasites.