δ34S for tracing the origin of cheese and detecting its authenticity

Casein δ³⁴S of 725 samples of cheese from all over the world were measured using IRMS. δ³⁴S alone made it possible to establish characteristic ranges of values for two types of Italian cheese (Grana Padano PDO and Parmigiano Reggiano PDO) and for the different regions and provinces of both the Grana Padano PDO and the Parmigiano Reggiano PDO zones. Moreover, δ³⁴S of PDO Italian samples were compared to both Italian (not PDO) and foreign competitive cheese samples. In all the cases, sulfur isotopic ratio analysis was a powerful tool to fix characteristic ranges of values for cheeses coming from different countries and to improve the information given by other isotopic parameters.     

Ripening and geographical characterization of Parmigiano Reggiano cheese by 1H NMR spectroscopy

Samples of Italian Parmigiano Reggiano cheese of different ripening stages (14, 24 and 30 months) were analyzed by (1)H NMR spectroscopy and the water-soluble metabolites content compared with samples of "Grana type" cheese from east Europe countries. Different multivariate statistical protocols were examined to build up a stable model for both ripening and geographical discrimination among the samples. The discriminant approach revealed a larger metabolite content increasing with ripening process; in particular younger samples were characterized by a higher content in leucine and isoleucine, while 30 months samples by a larger extent of threonine. The use of a classification approach resulted in a better discrimination of geographical samples. Foreign "Grana type" samples resulted well differentiated with respect to all Italian samples of Parmigiano Reggiano cheese. Foreign samples were characterized by a large amount of leucine and isoleucine, compounds that typified young Italian samples (14 months), and also by lactate, butanoate and acetate, thus suggesting short ripening for foreign samples. Parmigiano Reggiano samples were instead characterized by a large amount of all other compounds, in particular by threonine, which typified old samples (30 months), and other amino acids.     

Differentiation among dairy products by combination of fast field cycling NMR relaxometry data and chemometrics

A set of commercial milk and Sicilian cheeses was analysed by a combination of fast field cycling (FFC) nuclear magnetic resonance (NMR) relaxometry and chemometrics. The NMR dispersion (NMRD) curves were successfully analysed with a mathematical model applied on Parmigiano–Reggiano (PR) cheese. Regression parameters were led back to the molecular components of cheeses (water trapped in casein micelles, proteins and fats) and milk samples (water belonging to hydration shells around dispersed colloidal particles of different sizes and bulk water). The application of chemometric analysis on relaxometric data enabled differentiating milk from cheeses and revealing differences within the two sample groups of either cheeses or milk samples. Marked differences among cheeses were evidenced by statistical analysis of the sole quadrupolar peaks parameters, suggesting that these contain information on the nature of the milk used during cheese production. Hence, combination of FFC NMR and chemometrics represents a powerful tool to investigate alterations in dairy products.     

H,C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses

In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors.     

delta(13)C- and delta(18)O-values of glycerol of food fats

The average values of carbon and oxygen isotopic contents (delta(13)C and delta(18)O) of 36 glycerol samples from fats have been determined. The examined samples arise from many fats of animal and plant origin, as well as from the three Italian hard cheeses Parmigiano-Reggiano, Grana Padano and Trentingrana. The total (13)C content allows one to distinguish between glycerol from plants with the C-4 carbon fixation pathway (maize, mean delta(13)C = -14.4 per thousand) and that from plants with the C-3 pathway (mean delta(13)C = -30.7 per thousand). The delta(13)C-values of glycerols of animal origin seem to depend on the diet of the animal, as suggested by the mean values -29.6, -29.0 and -25.1 per thousand, respectively, observed for Parmigiano-Reggiano, Trentingrana and Grana Padano. Additionally, the mean total (18)O content of glycerol samples of vegetable origin is approximately 23.8 per thousand, while that from animal fat is 15.1 per thousand. However, the delta(18)O mean values relative to Parmigiano-Reggiano, Grana Padano and Trentingrana are 11.8, 16.0 and 13.8 per thousand, respectively. The combination of the (13)C and (18)O measurements relative to the fat glycerol of the three cheeses might be considered a potential criterion of authentication.     

Characterization of the volatile organic compounds of Italian 'Fossa' cheese by solid-phase microextraction gas chromatography/mass spectrometry

Fossa cheese is an Italian hard cheese, ripened for up to 3 months in underground pits dug into tuffaceous rock. During this period, the cheese develops a unique flavour and intense and somewhat piquant aroma. Solid-phase microextraction gas chromatography/mass spectrometry (SPME-GC/MS) was utilized to characterize the volatile organic compounds (VOCs) of Fossa cheese. A total of 75 VOCs were separated and identified; in particular, the major class of compounds found in the cheeses ripened in the pits were the esters of fatty acids. Discriminant analysis of volatile profiles allowed us to distinguish between cheeses in different stages of seasoning (60-day-old cheese and cheese ripened an additional 90 days in and out of the pits).     

Is high‐resolution magic angle spinning NMR a practical speciation tool for cheese samples? Parmigiano Reggiano as a case study

High-resolution magic angle spinning (HRMAS) NMR is probably the most apt NMR method to analyze complex materials involving a solid phase, e.g. foodstuffs. We present here an HRMAS analysis of grated cheese (Parmigiano Reggiano). A full NMR characterization of this cheese allows the identification of the presence of fatty acids (saturated and unsaturated), amino acids and other small organic molecules. Since the presence and relative concentration of these molecules have previously been shown to correlate with organoleptic, origin and age characterization, HRMAS NMR of cheese is likely to provide a good complimentary tool for the analysis of this food material.     

Evaluation of a new modified QuEChERS method for the monitoring of carbamate residues in high‐fat cheeses by using UHPLC–MS/MS

A simple and efficient method for the determination of 28 carbamates in high‐fat cheeses is proposed. The methodology is based on a modified quick, easy, cheap, effective, rugged, and safe procedure as sample treatment using a new sorbent (Z‐Sep⁺) followed by ultra‐high performance liquid chromatography with tandem mass spectrometry determination. The method has been validated in different kinds of cheese (Gorgonzola, Roquefort, and Camembert), achieving recoveries of 70–115%, relative standard deviations lower than 13% and limits of quantification lower than 5.4 μg/kg, below the maximum residue levels tolerated for these compounds by the European legislation. The matrix effect was lower than ±30% for all the studied pesticides. The combination of ultra‐high performance liquid chromatography and tandem mass spectrometry with this modified quick, easy, cheap, effective, rugged, and safe procedure using Z‐Sep⁺ allowed a high sample throughput and an efficient cleaning of extracts for the control of these residues in cheeses with a high fat content.     

N-磷酰化肽酯及小肽与溶菌酶相互作用的ESI-MS研究

用ESI-MS研究了一系列结构具有可比性的N-磷酰化肽酯及小肽和溶菌酶的非共价相互作用, 比较了磷酰化肽酯及小肽分子中的不同基团对相互作用的影响. 结果表明—OH对其与溶菌酶的相互作用有较大贡献; 芳香环由于位阻原因, 对相互作用有促进和阻碍双重效应; 当—OH与芳香环相连时会发生协同效应, 可使相互作用显著增强. 磷酰化肽酯及小肽的体积大小、空间位阻对相互作用亦有显著影响. 磷酰化二肽中氨基酸残基的构型、顺序、碳链长短的变化(增加1～2个C)对其与蛋白溶菌酶之间的相互作用在质谱中没有表现出影响. 分子结构较为伸展、分子柔顺性好、空间位阻较小的磷酰化小肽更容易使蛋白在溶液中的构象趋于收缩, 而构象较为收缩的蛋白分子更易结合空间位阻较小的磷酰化小肽分子.     

3-Nitro-l,2,4-triazol-l-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a condensing reagent for solid-phase peptide synthesis

Solid-phase oligopeptide synthesis has been well developed and most short oligopeptides can now be easily synthesized. However, when a desired oligopeptide forms a secondary structure or includes less reactive amino acids such as aminoisobutyric acid, its terminal amino groups become less reactive and synthesis of the desired oligopeptides becomes difficult. To expand the number of synthetic peptide sequences, we have developed efficient coupling conditions using 3-nitro-l,2,4-triazol-l-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a highly reactive condensing reagent on an unswellable solid support. PyNTP demonstrated higher reactivity than conventional condensing reagents and the optical purity of the synthesized oligopeptides was sufficiently high for application to general oligopeptide synthesis.     

Direct molecular weight determination of resin-bound oligopeptides using 252Cf plasma-desorption mass spectrometry.

Plasma-desorption mass spectrometry is proved to be capable of the direct molecular weight determination of totally protected resin-bound oligopeptides. The molecular weight measured is the molecular weight of the fully protected oligopeptide including the molecular weight of the linker, which connects the oligopeptide and the polystyrene resin. This method makes it possible to check the success of (a step in) the synthesis of a peptide before its deprotection or cleaving from the resin. This can make it a strong tool in the analysis of resin-bound oligopeptides.     

Pyridine-substituted oligopeptides as scaffolds for the assembly of multimetallic complexes: variation of chain length

This paper presents the synthesis and characterization of pyridine-substituted artificial oligopeptides with an aminoethylglycine backbone of varying length, which are designed to act as scaffolds for the self-assembly of multimetallic structures. The identities and purities of the oligopeptides are confirmed with mass spectrometry, (1)H NMR, HPLC, and pH titrations. The acid dissociation constants for the oligopeptides were determined and were found to decrease with increasing pyridine units. Titrations of the oligopeptides with Cu(II) and Pt(II) complexes containing the tridentate ligands 2,2':6',2'-terpyridine and pyridine 2,6-dicarboxylic acid were monitored using UV-visible absorption spectroscopy and showed stoichiometric binding based on the number of pyridines on the peptide strand. Metal titrations performed using an analogous oligopeptide with methyl substituents (in place of the pyridine ligands) showed very weak or no binding. In the case of the oligopeptides containing bound Pt(terpyridine)(2+) complexes, cyclic voltammetry reveals two sequential one-electron reductions at formal potentials that do not vary as a function of oligopeptide length. The measured diffusion coefficients were measured with chronoamperometry and were found to decrease with increasing oliopeptide length.     

Sequence analysis of derivatized peptides by high-performance liquid chromatography-mass spectrometry

N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Applications of HPLC-MS in compound Ilex pubescens extract study

In this paper, high performance liquid chromatography (HPLC) along with mass spectrometry (MS) and HPLC along with a diode array detector (DAD) was used to study the compound Ilex pubescens extract. Two ionization techniques: electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were used in this work. The liquid chromatograms obtained by DAD, total ion chromatograms (TIC) from positive-and negative-ion ESI-MS and the positive-and negative-ion APCI-MS were compared. The liquid chromatograms obtained by TIC from ESI-MS provided more information on chromatographic peaks than those obtained by DAD or TIC from APCI-MS. It is suggested that the fingerprints of the compound Ilex pubescens extract should be provided by the liquid chromatograms obtained by DAD together with TIC from the negative-ion ESI-MS. The molecular weights of the nine main components in an HPLC-DAD chromatogram were determined by the corresponding positive-and negative-ion ESI and the positive-and negative-ion APCI mass spectra information. In the liquid chromatogram obtained by TIC from the negative-ion ESI-MS, the molecular weights of 23 main components were determined based on the corresponding positive-and negative-ion ESI mass spectra information.     

Racemic beta-sheets as templates for the generation of homochiral (isotactic) peptides from aqueous solutions of (RS)-valine or -leucine N-carboxy- anhydrides: relevance to biochirogenesis

As part of our program on biochirogenesis of homochiral peptides from racemic precursors, we report the feasibility of obtaining peptides with homochiral sequences composed of up to 25 residues of the same handedness in the polymerization of racemic valine or leucine N-carboxyanhydrides in aqueous solutions, as initiated by amines. The composition of the oligopeptides was determined by MALDI-TOF mass spectrometry, and the sequences of some of the heterochiral diastereoisomers were studied by MALDI-TOF MS/MS performed on samples in which the S enantiomers of the monomer were tagged with deuterium atoms. The process comprises several steps: 1) a Markov mechanism of asymmetric induction in the early stages of the polymerization yields libraries of racemic oligopeptides enriched with isotactic diastereoisomers, together with oligopeptide sequences containing enantiomeric blocks of homochiral residues; 2) the short peptides self-assemble into racemic colloidal architectures that serve as regio-enantioselective templates in the ensuing process of chain elongation; 3) homochiral residues of the amino acids located at the periphery of these colloidal aggregates exert efficient enantioselection, which results in the formation of long isotactic oligopeptides. The final diastereoisomeric distribution of the peptides depends upon the composition of the templates, which is determined by the concentration of the initiator. The racemic mixtures of isotactic peptides can be desymmetrized by using enantiopure methyl esters of alpha-amino acids as initiators.     

Identification of peptides in antimicrobial fractions of cheese extracts by electrospray ionization ion trap mass spectrometry coupled to a two-dimensional liquid chromatographic separation

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled to a two-dimensional liquid chromatographic separation was applied to the identification of peptides in antimicrobial fractions of the aqueous extracts of nine Italian cheese varieties. In particular, the chromatographic fractions collected during a preliminary fast protein liquid chromatography (FPLC) separation on the cheese extracts were assayed for antimicrobial activity towards Lactobacillus sakei A15. Active fractions were subsequently analyzed by reversed-phase high-performance liquid chromatography electrospray ionization sequential mass spectrometry (HPLC/ESI)-ITMSn, with n up to 3. Peptide identification was then performed starting from a conventional proteomics approach based on tandem mass spectrometric (MS/MS) analysis followed by database searching. In many cases this strategy had to be integrated by a careful correlation between spectral information and predicted peptide fragmentation, in order to reach unambiguous identifications. When even this integrated approach failed, MS3 measurements provided decisive information on the amino acid sequence of some peptides, through fragmentation of pendant groups along the peptide chain. As a result, 45 peptides, all arising from hydrolysis of milk caseins, were identified in nine antimicrobial FPLC fractions of aqueous extracts obtained from five of the nine cheese varieties considered. Many of them corresponded to peptides already known to exhibit biological activity.     

Comparison of direct infusion and on-line liquid chromatography/electrospray ionization mass spectrometry for the analysis of nucleic acids

The applicability of ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) and direct infusion/ESI-MS to the characterization of nucleic acid mixtures was evaluated by the analysis of the reaction products obtained from solid-phase synthesis of a 39-mer oligonucleotide. IP-RP-HPLC/ESI-MS was performed using 200 microm i.d. capillary columns packed with octadecylated, micropellicular poly(styrene-divinylbenzene) particles and applying gradients of acetonitrile in 50 mM triethylammonium bicarbonate (TEAB). Three different solvent systems were utilized for direct infusion/ESI-MS with removal of metal cations by on-line cation exchange: (1) 10 mM triethylamine (TEA) in 50% aqueous acetonitrile, (2) 2.2 mM TEA, 400 mM hexafluoro-2-propanol (HFIP) in 20% aqueous methanol and (3) 50 mM TEAB in 10% aqueous acetonitrile. Owing to its separation capability, the highest selectivity and specificity were achieved with IP-RP-HPLC/ESI-MS, which, apart form the 39-mer target sequence, allowed the identification of two isobutyryl-protected target sequences and a 10-mer and 20-mer failure sequence. Direct infusion/ESI-MS with TEA-acetonitrile or TEA-HFIP-methanol as solvent revealed signals for the 39-mer in the m/z range 700-1600. The presence of derivatives containing one, two, three and four isobutyryl groups indicated that the hydrolysis of the protecting groups after solid-phase synthesis was not complete. Failure sequences could not be identified by direct infusion/ESI-MS under conditions favoring multiple charging of the analytes owing to the high chemical background and coincidental overlapping of m/z signals. However, efficient charge state reduction upon addition of carbonic acid to the electrosprayed solvent shifted the signals of the 39-mer and derivatives to m/z values >2400 and allowed the detection of seven different failure sequences, ranging from the 8-mer to the 23-mer, in the mixture.     

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 sheep's milk to goat's cheeses, because it has a very similar taste to goat's milk, but is more available, and is commonly considered to have a better capacity to curdle. For similar reasons, and due to economic convenience, sheep's cheeses may also contain fraudulent cow's milk. In order to detect this fraud, an EU official method may be used, but it is only a qualitative method (presence/absence of cow's milk). A method able to quantify the presence of sheep's milk during cheese production in goat's and cow's 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 sheep's peptide produced by plasmin hydrolysis (m/z = 860) was accurately selected and analyzed to understand if, and by how much, a declared pure goat's cheese contains sheep's milk. By analyzing the same peptide it is also possible to detect if, and by how much, a declared pure sheep's milk contains, or not, cow's milk. The method was applied to several goat's and cow's cheese samples. Quantitation was performed with a calibration curve obtained by analyzing curd cheeses containing different percentages of sheep's milk. The method detection limit and method quantitation limit were evaluated. This method appears accurate and suitable for detecting up to 2% of sheep's milk in cheeses. Copyright © 2010 John Wiley & Sons, Ltd.     

Sequence-specific fragmentation from oligopeptides using plasma-desorption mass spectrometry

252Cf plasma-desorption mass spectrometry (PDMS) has been demonstrated to provide sequence-specific fragmentation for several oligopeptides. The nature of the fragment ions observed is generally similar to that observed using liquid secondary-ion mass spectrometry (LSIMS) and can be observed using less sample than LSIMS requires, but PDMS spectra are acquired at a lower resolution. In addition, the molecular weight of some of the oligopeptides studied exceeds that which is generally accepted as within the sequence range of LSIMS. The specific series of sequence ions that predominate in the PDMS spectra appear to be related to the amino acid compositions and sequences of the oligopeptides.