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Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended. [ABSTRACT FROM AUTHOR]

Most gluten-reduced beers are produced using an enzyme called proline endopeptidase (PEP), which proteolyzes the gluten by cleaving at proline residues. However, the gluten content of beers brewed in the presence of PEP cannot be verified since current analytical methods are not able to accurately quantitate gluten in fermented foods. In this work, mass spectrometry was used to qualitatively characterize the gluten in a wheat-gluten-incurred sorghum model beer brewed with and without the addition of PEP. Hydrolyzed gluten peptides and chymotryptic gluten peptides produced from intact gluten proteins were detected in beer brewed in the presence of up to 6 times the manufacturer's recommended dosage of PEP. The observation of chymotryptic gluten peptides indicates that some gluten proteins remained, at least partially, intact after fermentation and enzymatic treatment. Less intact gluten was observed in beer brewed in the presence of PEP, but more hydrolyzed gluten peptides were consequently observed in PEP-containing beer. Gluten peptides that contained immunogenic sequences known to be associated with celiac disease were detected in PEP-containing beer. [ABSTRACT FROM AUTHOR]

The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation. The most notable example is the use of deuteroacetylation to quantitate lysine acetylation. In this work, levels of lysine ubiquitination were quantitated using a structurally homologous label that is chemically similar to the diglycine (GlyGly) tag, which is left at the ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically equivalent monoglycine (Gly) tag that is analogous to the corresponding GlyGly tag proved that the monoglycine tag can be used for the quantitation of ubiquitination. A glycinylation protocol was then established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. Ubiquitin multimers were used to show that after glycinylation and tryptic digestion, the mass spectrometric response from the corresponding analogous tagged peptides could be compared for relative quantitation. For a proof of principle regarding the applicability of this technique to the analysis of ubiquitination in biological samples, the glycinylation technique was used to quantitate the increase in monoubiquitinated histone H2B that is observed in yeast which lacks the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast. [ABSTRACT FROM AUTHOR]

The core histones, H2A, H2B, H3 and H4, undergo post-translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono-, di- and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high-performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom-up liquid chromatography-mass spectrometric analysis. The deuteroacetylation of unmodified or mono-methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification 'cross-talk' by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]