A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

In vitro digestion of nitrite and nitrate preserved fermented sausages - New understandings of nitroso-compounds' chemical reactivity in the digestive tract.

In vitro digestions of dry-cured sausages formulated with four different rates of added sodium nitrite and sodium nitrate (NaNO2 / NaNO3, in ppm: 0/0; 80/80; 120/120; 0/200) were performed with a dynamic gastrointestinal digester (DIDGI®). The chemical reactivity of the potentially toxic nitroso-compounds (NOCs), oxidation reactions products and different iron types were evaluated over time. No nitrite nor nitrate dose effect was observed on NOCs' chemical reactivity. Nitrosothiols were scarce, and nitrosylheme was destabilized for every conditions, possibly leading to free iron release in the digestive tract. Total noN-volatile N-nitrosamines concentrations increased in the gastric compartment while residual nitrites and nitrates remained stable. The minimal rate of 80/80 ppm nitrite/nitrate was enough to protect against lipid oxidation in the digestive tract. The present results provide new insights into the digestive chemistry of dry sausages, and into new reasonable arguments to reduce the load of additives in formulations.

Digestomic data of proteolysis during whether post rumen digestion after tannin supplementation.

The protein degradation of alfalfa hay after tannin supplementation was monitored during wethers digestion. Three rumen-cannulated wethers were infused a tannin solution, and water for control, through the cannula. The digestion time-points samples were collected in vivo in the rumen and in vitro in the abomasum, and the small intestine compartments. The digestomic dataset was acquired by identifying and quantifying the peptides resulting from the protein degradation, using high-resolution LC-MS/MS mass spectrometry and label-free quantitation. The digestomic dataset is the compilation of proteomic data acquired in the rumen and peptidomic data acquired in the abomasum and in the small intestine. The proteomic analysis identified 20 Medicago proteins in the rumen fluid, based on 169 peptides of which 140 are unique. The peptidomic analysis identified 28 Medicago proteins in the abomasum, based on 575 peptides of which 363 are unique, and 11 Medicago proteins in the small intestine, based on 94 peptides of which 63 are unique. This digestomic dataset of proteolysis during sheep post rumen digestion after tannin supplementation reveals the protein regions protected by tannin supplementation, and could be reused in studies related to the protein use efficiency by ruminants.

Impact of Tannin Supplementation on Proteolysis during Post-Ruminal Digestion in Wethers Using a Dynamic In Vitro System: A Plant (Medicago sativa) Digestomic Approach.

The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.

Design of an In Vitro Model to Screen the Chemical Reactivity Induced by Polyphenols and Vitamins during Digestion: An Application to Processed Meat.

Processed meats' nutritional quality may be enhanced by bioactive vegetable molecules, by preventing the synthesis of nitrosamines from N-nitrosation, and harmful aldehydes from lipid oxidation, through their reformulation. Both reactions occur during digestion. The precise effect of these molecules during processed meats' digestion must be deepened to wisely select the most efficient vegetable compounds. The aim of this study was to design an in vitro experimental method, allowing to foresee polyphenols and vitamins' effects on the chemical reactivity linked to processed meats' digestion. The method measured the modulation of end products formation (specific nitroso-tryptophan and thiobarbituric acid reactive substances (TBARS)), by differential UV-visible spectrophotometry, according to the presence or not of phenolic compounds (chlorogenic acid, rutin, naringin, naringenin) or vitamins (ascorbic acid and trolox). The reactional medium was supported by an oil in water emulsion mimicking the physico-chemical environment of the gastric compartment. The model was optimized to uphold the reactions in a stable and simplified model featuring processed meat composition. Rutin, chlorogenic acid, naringin, and naringenin significantly inhibited lipid oxidation. N-nitrosation was inhibited by the presence of lipids and ascorbate. This methodology paves the way for an accurate selection of molecules within the framework of processed meat products reformulation.

Determination of nitroso-compounds in food products.

Nitrite and nitrate are present in many foods. Nitrate can be converted into nitrite in human body. Nitrite can react with secondary amines to form secondary amines and with thiols to form nitrosothiols. Some nitrosamines are cancers suspect. Because of their importance in terms of human health, research on these compounds is still topical and the use of a rapid and reproducible method for determination and quantification of these compounds is necessary. This article presents a method to study the chemical reactivity of nitrite in meat products through the analysis of non-volatile nitrosamines and nitrosothiols based on:•A specific alkaline and heat extraction of nitro-compounds followed by deprotenization by ultrafiltration•NO detection by the Griess reaction•NO released from S-NO and N-NO bonds by UV light followed by a specific cleavage of S-NO bonds with HgCl2This method, validated on cured meat products, could be developed in the same way on all products containing nitrite and nitrate and leading to the formation of nitroso-compounds. The limit of detection for these compounds are of the order of the micromole per liter.

Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.

Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment.

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

Protein Profile and Simulated Digestive Behavior of Breast Milk from Overweight and Normal Weight Mothers.

Human milk proteins have shown to vary in concentration and distribution through lactation. However, while some regulatory components, such as hormones, have shown associations with regard to the mothers' body mass index, there is limited information on the possible influence of this condition on the whole protein distribution. The objective of this study was to evaluate the protein profile of human milk from normal weight and overweight or obese mothers to identify differences in protein expression in colostrum, transitional and mature milk. The mass spectrometry analysis showed the ability to class with a high degree of confidence the lactation state and the milk profile according to the mother's condition. Individual milk samples were subjected to a digestion in vitro model that takes into account the specificities of the gastrointestinal conditions of full-term newborn infants. The digestion products were compared with available data from the digestive contents in newborns. The behavior of the most abundant proteins and the overall peptide generation and survival, showed good correspondence with in vivo data.

New Insights into the Chemical Reactivity of Dry-Cured Fermented Sausages: Focus on Nitrosation, Nitrosylation and Oxidation.

Nitrite and nitrate are added to cured meat for their bacteriological, technological and sensorial properties. However, they are suspected to be involved in the formation of nitroso compounds (NOCs), such as potentially mutagenic nitrosamines, nitrosylheme and nitrosothiols. Controlling the sanitary and sensorial qualities of cured meat products by reducing these additives requires elucidating the mechanisms involved in the formation of NOCs. To this end, we studied the dose-response relationship of added sodium nitrite and/or sodium nitrate (0/0, 80/80, 0/200, and 120/120 ppm) on the formation of NOCs in dry cured fermented sausages. The results showed a basal heme iron nitrosylation in the absence of NaNO2/NaNO3 due to starter cultures. This reaction was promoted by the addition of NaNO2/NaNO3 in the other conditions. Reducing the dose to 80/80 ppm still limits lipid oxidation without the formation of non-volatile nitrosamines. Conversely, the addition of NO2/NO3 slightly increases protein oxidation through higher carbonyl content. The use of 80/80 ppm could be a means of reducing these additives in dry-cured fermented meat products.

Chemical reactivity of nitrite and ascorbate in a cured and cooked meat model implication in nitrosation, nitrosylation and oxidation.

Nitrite, added to cured meat for its bacteriological and technological properties, is implicated in the formation of nitroso compounds (NOCs), such as nitrosylheme, nitrosamines and nitrosothiols, suspected to have a potential impact on human health. The mechanisms involved in NOC formation are studied in regard with the dose-response relationship of added nitrite and its interaction with ascorbate on NOC formation in a cured and cooked meat model. The impact of a second cooking stage on nitrosation was evaluated. The addition of nitrite in the cured and cooked model promoted heme iron nitrosylation and S-nitrosation but not N-nitrosation. Nitrite reduced lipid oxidation without an additional ascorbate effect. The second cooking sharply increased the nitrosamine content while the presence of ascorbate considerably lowered their levels and protected nitrosothiols from degradation. This study gives new insights on the chemical reactivity of NOCs in a cured meat model.

Next-Generation Sequencing and MALDI Mass Spectrometry in the Study of Multiresistant Processed Meat Vancomycin-Resistant Enterococci (VRE).

Vancomycin-resistant enterococci (VRE), due to their intrinsic resistance to various commonly used antibiotics and their malleable genome, make the treatment of infections caused by these bacteria less effective. The aims of this work were to characterize isolates of Enterococcus spp. that originated from processed meat, through phenotypic and genotypic techniques, as well as to detect putative antibiotic resistance biomarkers. The 19 VRE identified had high resistance to teicoplanin (89%), tetracycline (94%), and erythromycin (84%) and a low resistance to kanamycin (11%), gentamicin (11%), and streptomycin (5%). Based on a Next-Generation Sequencing NGS technique, most isolates were vanA-positive. The most prevalent resistance genes detected were erm(B) and aac(6')-Ii, conferring resistance to the classes of macrolides and aminoglycosides, respectively. MALDI-TOF mass spectrometry (MS) analysis detected an exclusive peak of the Enterococcus genus at m/z (mass-to-charge-ratio) 4428 ± 3, and a peak at m/z 6048 ± 1 allowed us to distinguish Enterococcus faecium from the other species. Several statistically significant protein masses associated with resistance were detected, such as peaks at m/z 6358.27 and m/z 13237.3 in ciprofloxacin resistance isolates. These results reinforce the relevance of the combined and complementary NGS and MALDI-TOF MS techniques for bacterial characterization.

Putative Protein Biomarkers of Escherichia coli Antibiotic Multiresistance Identified by MALDI Mass Spectrometry.

The commensal bacteria Escherichia coli causes several intestinal and extra-intestinal diseases, since it has virulence factors that interfere in important cellular processes. These bacteria also have a great capacity to spread the resistance genes, sometimes to phylogenetically distant bacteria, which poses an additional threat to public health worldwide. Here, we aimed to use the analytical potential of MALDI-TOF mass spectrometry (MS) to characterize E. coli isolates and identify proteins associated closely with antibiotic resistance. Thirty strains of extended-spectrum beta-lactamase producing E. coli were sampled from various animals. The phenotypes of antibiotic resistance were determined according to Clinical and Laboratory Standards Institute (CLSI) methods, and they showed that all bacterial isolates were multi-resistant to trimethoprim-sulfamethoxazole, tetracycline, and ampicillin. To identify peptides characteristic of resistance to particular antibiotics, each strain was grown in the presence or absence of the different antibiotics, and then proteins were extracted from the cells. The protein fingerprints of the samples were determined by MALDI-TOF MS in linear mode over a mass range of 2 to 20 kDa. The spectra obtained were compared by using the ClinProTools bioinformatics software, using three machine learning classification algorithms. A putative species biomarker was also detected at a peak m/z of 4528.00.

Using a dynamic artificial digestive system to investigate heme iron nitrosylation during gastro-intestinal transit.

The International Agency for Research on Cancer recently classified cured meats as carcinogenic for humans and red meats as probably carcinogenic. Mutagens can be formed during meat process or digestion. In a previous study, we used a dynamic artificial digestive system (DIDGI®) to investigate protein oxidation and N-nitrosation during bovine meat digestion. This new paper completes the previous one by focusing on the endogenous heme iron nitrosylation. Low nitrosylation due to nitrate initially present in meat and to ammonia oxidation in the stomach was observed in the digestive tract even in conditions in which no nitrite was added to the model. The endogenous addition of nitrite (1 mM) considerably increased heme iron nitrosylation while a significant decrease was observed with prior meat cooking (30 min at 60 and 90 °C).

Deciphering PSE-like muscle defect in cooked hams: A signature from the tissue to the molecular scale.

PSE-like technological defect in the meat industry is of great importance due, to the economic loss it can cause. It has been studied from the biochemical perspective but very few studies have focused on tissular characterization. This study proposes innovative approaches that combine mechanistic elucidation and the discovery of potential biomarkers. This study focused on muscle destructuration using imaging and label-free quantitation. Oxidative stress and apoptotic processes were found to be linked to the specific evolution of the PSE-like destructuration zone, namely 'inner', within hams. Four m/z values were found to be related to the specific localization of the PSE-like defect, and a mass shift of 27 Da suggested a possible connection with oxidation. These potential markers of the PSE-like area in ham provide a new perspective to sort raw material based on the possible development of PSE-like areas.

To what extent does the nitrosation of meat proteins influence their digestibility?

Nitrosation can occur during meat digestion due to the physicochemical conditions of the stomach (low pH and reducing conditions). The aim of the present study was to elucidate the link between the nitrosation of proteins from beef meat and their digestibility by comparing cooked meat digested with and without the addition of nitrite. To do this, a dynamic in vitro artificial digestive computer-controlled system (DIDGI®) was used to reflect human gastro-intestinal conditions. Peptides and proteins from gastrointestinal digestion were identified by high-resolution LC-MS/MS mass spectrometry. The results showed a dynamic digestion pattern of meat proteins according to their cellular localization. A combined effect of the digestive compartment and the addition of nitrite was established for the first time on peptides profile, linking the nitrosation of proteins and their digestibility.

MALDI mass spectrometry imaging and in situ microproteomics of Listeria monocytogenes biofilms.

MALDI-TOF Mass spectrometry Imaging (MSI) is a surface-sampling technology that can determine spatial information and relative abundance of analytes directly from biological samples. Human listeriosis cases are due to the ingestion of contaminated foods with the pathogenic bacteria Listeria monocytogenes. The reduction of water availability in food workshops by decreasing the air relative humidity (RH) is one strategy to improve the control of bacterial contamination. This study aims to develop and implement an MSI approach on L. monocytogenes biofilms and proof of concept using a dehumidified stress condition. MSI allowed examining the distribution of low molecular weight proteins within the biofilms subjected to a dehumidification environment, mimicking the one present in a food workshop (10 °C, 75% RH). Furthermore, a LC-MS/MS approach was made to link the dots between MSI and protein identification. Five identified proteins were assigned to registered MSI m/z, including two cold-shock proteins and a ligase involved in cell wall biogenesis. These data demonstrate how imaging can be used to dissect the proteome of an intact bacterial biofilm giving new insights into protein expression relating to a dehumidification stress adaptation. Data are available via ProteomeXchange with identifier PXD010444. BIOLOGICAL SIGNIFICANCE: The ready-to-eat food processing industry has the daily challenge of controlling the contamination of surfaces and machines with spoilage and pathogenic microorganisms. In some cases, it is a lost cause due to these microorganisms' capacity to withstand the cleaning treatments, like desiccation procedures. Such a case is the ubiquitous Gram-positive Bacterium Listeria monocytogenes. Its surface proteins have particular importance for the interaction with its environment, being important factors contributing to adaptation to stress conditions. There are few reproducibly techniques to obtain the surface proteins of Gram-positive cells. Here, we developed a workflow that enables the use of MALDI imaging on Gram-positive bacterium biofilms to study the impact of dehumidification on sessile cells. It will be of the most interest to test this workflow with different environmental conditions and potentially apply it to other biofilm-forming bacteria.

Iron-catalysed chemistry in the gastrointestinal tract: Mechanisms, kinetics and consequences. A review.

Chemical changes that occur during the storage and processing of food can affect its nutritional content. During digestion, the exposure of food to considerable variations of pH and high oxygen and peroxide concentrations also participates in the deterioration of nutrients, with a negative impact on the nutritional value of the diet and harmful consequences for human health. Iron plays a key role in gastrointestinal chemistry. Haem iron, which exists only in meat, and non-haem iron, present in most foods, are catalysts of most of the reactions implicated in the deterioration of nutrients. Disintegration of food matrix due to mechanical forces and enzymatic hydrolysis favour this endogenous process. This paper provides a review of what is known in the literature concerning the mechanisms and kinetics of endogenous reactions catalysed by iron. The main consequences on nutrient bioavailability are reported and protective strategies against the deleterious effect of iron are discussed.

Myofiber metabolic type determination by mass spectrometry imaging.

Matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a powerful tool that opens new research opportunities in the field of biology. In this work, predictive model was developed to discriminate metabolic myofiber types using the MALDI spectral data. Rat skeletal muscles are constituted of type I and type IIA fiber, which have an oxidative metabolism for glycogen degradation, and type IIX and type IIB fiber which have a glycolytic metabolism, present in different proportions according to the muscle function and physiological state. So far, myofiber type is determined by histological methods that are time consuming. Thanks to the predictive model, we were able to predict not only the metabolic fiber type but also their location, on the same muscle section that was used for MALDI imaging. Copyright © 2017 John Wiley & Sons, Ltd.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF.

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.