Comparison of allergen quantification strategies for egg, milk, and peanut in food using targeted LC-MS/MS.

Methods for the detection and quantification of food allergens in complex matrices are necessary to ensure compliance with labeling regulations and assess the effectiveness of food allergen preventive controls. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as an orthogonal technique in complement to immunochemical-based assays. However, the absence of established guidelines for MS-based quantification of allergens in food has limited harmonization among the method development community. In this study, different quantification strategies were evaluated using a previously developed multiplexed LC-MS/MS method for the detection of egg, milk, and peanut. Peptide performance criteria (retention time, signal-to-noise ratio, and ion ratio tolerance) were established and quantification approaches using varying calibrants, internal standards, background matrices, and calibration curve preparation schemes were systematically evaluated to refine the previous method for routine laboratory use. A matrix-matched calibration curve using allergen ingredients as calibrants and stable isotope-labeled peptides as internal standards provided the most accurate quantitative results. The strategy was further verified with commercially available reference materials and allowed for the confident detection and quantification of food allergens. This work highlights the need for transparency in calibration strategy and peptide performance requirements for effective evaluation of mass spectrometric methods for the quantification of food allergens.

Detection of gluten in a pilot-scale barley-based beer produced with and without a prolyl endopeptidase enzyme.

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.

Analysis of Gluten in a Wheat-Gluten-Incurred Sorghum Beer Brewed in the Presence of Proline Endopeptidase by LC/MS/MS.

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.

Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

Characterization of grain-specific peptide markers for the detection of gluten by mass spectrometry.

Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1-100,000 ppm and analyzed to determine the lowest concentration at which the wheat "contaminant" could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats.

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants.

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.

Using glycinylation, a chemical derivatization technique, for the quantitation of ubiquitinated proteins.

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.

Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

A new resource-constrained triage method applied to victims of penetrating injury.

BACKGROUND: Resource-constrained triage occurs when the number of trauma patients exceeds the capacity for simultaneous transport and treatment. The objective of this article is to apply a new resource-constrained triage method (denoted Sacco triage method [STM]) to victims of penetrating trauma and compare it with existing methods. METHODS: STM is a mathematical model of resource-constrained triage. Its objective is to maximize expected survivors given constraints on the timing and availability of resources. The model incorporates estimates of time-dependent victim survival probabilities based on initial assessments and expected deteriorations. For application to penetrating trauma, an "RPM" score based on respiratory rate, pulse rate, and best motor response was used to predict survivability. Logistic function-generated survival probability estimates for scene values of RPM were determined from 7,274 penetrating injury patients from the Pennsylvania Trauma Outcome Study. The Delphi Method provided expert consensus on victim deterioration rates, and the model was solved using linear programming. The accuracy of predicting survivability was assessed using calibration and discrimination statistics. STM was compared with START (Simple Triage and Rapid Treatment)-like triage methods with respect to process and outcomes (assessed by expected number of survivors in simulated resource-constrained casualty incidents). RESULTS: RPM was shown to be an accurate predictor of survival probability for penetrating trauma, equivalent to the Revised Trauma Score and exceeding that of the Injury Severity Score, as measured by calibration and discrimination statistics. In the simulations, STM had substantially more expected survivors than did current triage methods. CONCLUSIONS: Resource-constrained triage is modeled as an evidence-based, outcome-driven method (STM) that maximizes expected survivors in consideration of resources. STM offers lifesaving and operational advantages over current methods.

Precise formulation and evidence-based application of resource-constrained triage.

OBJECTIVES: To develop a precise mathematical formulation of resource-constrained triage, denoted the Sacco triage method (STM), to develop an evidence-based application to blunt trauma, and to compare the STM with the simple triage and rapid treatment (START) method. METHODS: Resource-constrained triage is modeled mathematically as a classic resource allocation problem. The objective is to maximize expected survivors given constraints on the timing and availability of resources. The model incorporates estimates of time-dependent victim survival probabilities based on an initial assessment and expected deterioration. For application to blunt trauma, an "RPM" score, based on respiratory rate, pulse rate, and motor response, was used to predict survivability. Logistic function-generated survival probability estimates for scene values of RPM were determined from 76,459 blunt-injured patients from the Pennsylvania Trauma Outcome Study (PTOS). The Delphi method provided expert consensus on victim deterioration rates, and the model was solved using linear programming. STM was compared with START across various criteria of process and outcome. Outcome was measured by expected number of survivors in simulated resource-constrained casualty incidents. RESULTS: In this mathematical simulation, RPM was a more accurate predictor of survivability from blunt trauma than the Injury Severity Score and the Revised Trauma Score, as measured by calibration and discrimination statistics. STM resulted in greater expected survivorship than START in all simulations. CONCLUSIONS: Resource-constrained triage is modeled precisely as an evidence-based, outcome-driven method that maximizes expected survivors in consideration of resources. The lifesaving potential and operational advantages over current methods warrant scrutiny and further research.