A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using ...on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5
mg
L
−1.
Grape berries were classified according to diameter and total soluble solids (TSS) to study the effect of light and temperature on methoxypyrazines (MPs), glutathione (GSH), and hydroxycinnamates ...(HCAs) during the ripening of Sauvignon blanc. The light exposure of the fruiting zone was modified within leaf and lateral removal at the phenological stage berry of peppercorn size and no removal (control). In comparison to the control, the concentration of 3-isobutyl-2-methoxypyrazine (IBMP) was below the limit of detection in leaf removal 2 weeks before harvest. Leaf removal had no significant influence on GSH and HCAs in the grape juice at harvest. Berry diameter significantly influenced the concentration of IBMP in the grape juice and did not influence the concentration of GSH and HCAs. At harvest, the concentrations of IBMP in grape juices of similar TSS in the control were 12.6 and 5.2 ng/L in 15.5 and 13.5 mm berry diameter classes, respectively. Furthermore, the study showed that berries of the same diameter were not at the same physiological ripening level (not the same TSS).
Varietal thiol precursors in grapes are subject to metabolic changes during post-harvest treatments. Metabolic activity should therefore be limited after sampling to understand their biosynthesis in ...the berry and genetic regulation. In this study, berries were frozen in liquid nitrogen immediately after harvesting, transported in dry ice, stored briefly at -80 °C, cryo-milled and extracted without being thawed in cold methanol in a ratio of 1:4 (w/v). A UHPLC-MS/MS method for quantitative determination of the thiol precursors 3-S-glutathionylhexan-1-ol (G3MH), 3-S-cysteinylhexan-1-ol (Cys3MH), 4-S-glutathionyl-4-methylpentan-2-one (G4MMP) and 4-S-cysteinyl-4-methylpentan-2-one (Cys4MMP), glutathione, oxidized glutathione and L-methionine in grapes was developed. Reference material was provided through synthesis of precursors and their deuterium labelled analogues. The average thiol precursor content in grapes in 2013-15 was in the range 8-16 μg kg
for G3MH, 1-6 μg kg
for Cys3MH, 1-4 μg kg
for Cys4MMP and 0.3 μg kg
for G4MMP. In 2013 and 2014, the highest precursor content in mature Sauvignon Blanc grapes from vineyards located in Italy regarded G3MH, followed by Cys3MH, Cys4MMP and G4MMP. In 2015, G3MH was again the most abundant precursor, but followed by Cys4MMP, Cys3MH and G4MMP.
The capability of near infrared (NIR) spectroscopy was examined for the purposes of quality control of the traditional Slovenian dry-cured ham “
Kraški pršut.” Predictive models were developed for ...moisture, salt, protein, non-protein nitrogen, intramuscular fat and free amino acids in
biceps femoris muscle (n
=
135). The models' quality was assessed using statistical parameters: coefficient of determination (R
2) and standard error (se) of cross-validation (CV) and external validation (EV). Residual predictive deviation (RPD) was also assessed. Best results were obtained for salt content and salt percentage in moisture/dry matter (R
CV
2
>
0.90, RPD
>
3.0), it was satisfactory for moisture, non-protein nitrogen, intramuscular fat and total free amino acids (R
CV
2
=
0.75–0.90, RPD
=
2.0–3.0), while not so for protein content and proteolysis index (R
CV
2
=
0.65–0.75, RPD
<
2.0). Calibrations for individual free amino acids yielded R
CV
2 from 0.40 to 0.90 and RPD from 1.3 to 2.9. Additional external validation of models on independent samples yielded comparable results. Based on the results, NIR spectroscopy can replace chemical methods in quality control of dry-cured ham.
The aim of this study was to characterize the chemical profile of dry-cured ham selected and processed in accordance with the Slovenian consortium rules for »Kraški pršut« and to investigate the ...differences between the Biceps femoris (BF) and the Semimembranosus (SM) muscle. The free amino acid content, moisture, salt, protein, total nitrogen, non-protein nitrogen, proteolysis index, intramuscular fat and dry matter were analysed in 135 samples of dry-cured hams including both muscles. A modified method for sample preparation was developed for determination of free amino acids in dry-cured ham. The method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation parameters the method is appropriate for the determination of free amino acid content in dry-cured ham. Higher content for several free amino acids and total free amino acids were determined in the BF muscle compared to the SM muscle.
An extraction procedure for the aroma compounds from musts and wines has been developed, using solid-phase microextraction(DVB/CAR/PDMS fibre) from the headspace of heated samples (50 degree C). ...Analysis was performed withgas chromatography - mass spectrometry. The method was applicable to the analysis of different aroma compounds(aliphatic, aromatic aldehydes, terpenes) in a broad concentration range (1-5000 mu g L super(-1)). A stir-bar sorptive extractionprocedure was also tested, but was not effective enough due to the lack of a suitable desorption device. Free aroma compoundsin must samples of different grape varieties were analysed, and their release was evaluated after enzymatic oracidic hydrolysis. Different hydrolytic approaches were tested and the most successful was the enzymatic hydrolysis(with two different enzymes) and acidic hydrolysis at pH 3. Acidic hydrolysis at pH 1 resulted in substantial decompositionand re-arrangement reactions of terpenes. Non-hydrolysed terpene glycosides were extracted from the musts usingsolid-phase extraction and the extract was analysed with liquid chromatography - mass spectrometry (electrosprayinterface). Some compounds were tentatively identified as terpene glycosides.
For mass spectrometry-based diagnostics of microorganisms, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used to identify urinary ...tract pathogens. However, it requires a lengthy culture step for accurate pathogen identification, and is limited by a relatively small number of available species in peptide spectral libraries (≤3329). Here, we propose a method for pathogen identification that overcomes the above limitations, and utilizes the MALDI-TOF/TOF MS instrument. Tandem mass spectra of the analyzed peptides were obtained by chemically activated fragmentation, which allowed mass spectrometry analysis in negative and positive ion modes. Peptide sequences were elucidated de novo, and aligned with the non-redundant National Center for Biotechnology Information Reference Sequence Database (NCBInr). For data analysis, we developed a custom program package that predicted peptide sequences from the negative and positive MS/MS spectra. The main advantage of this method over a conventional MALDI-TOF MS peptide analysis is identification in less than 24 h without a cultivation step. Compared to the limited identification with peptide spectra libraries, the NCBI database derived from genome sequencing currently contains 20,917 bacterial species, and is constantly expanding. This paper presents an accurate method that is used to identify pathogens grown on agar plates, and those isolated directly from urine samples, with high accuracy.
Rationale
Selective derivatization of peptide N‐terminus with 4‐formyl‐benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge ...reduction conditions. Overlapped positive and negative tandem mass spectra show b‐ions making the assignment of b‐ion series fragments easy and accurate.
Methods
We developed an FBSA–peptide microwave‐assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non‐tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high‐quality data set of sulfonated b‐ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b‐ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y‐ions in positive tandem mass spectra to identify complete peptide sequences.
Results
The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high‐intensity signals of b‐ and y‐ions) compared to commonly used N‐terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b‐ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive‐ and negative ion modes, respectively. High visibility of b‐ion series in negative ion mode can be attributed to N‐terminal sulfonation that had no negative effect on the production of b‐ and y‐ion series in positive ion mode.
Conclusions
The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b‐ions in positive‐ and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.
PDF
