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In this work, we carry out the study of heavy flavor pentatuarks with four heavy quarks, which have typical $QQQQ\bar q$ configuration. Within the Chromomagnetic Interaction model, the mass spectrum of these discussed $QQQQ\bar q$ pentaquarks is given. In addition to the mass spectrum analysis, we also illustrate their two-body strong decay behavior by estimating some ratios of decay channels. By these effort, we suggest that future experiment should pay attention to this kind of pentaquark., 16 pages, 2 figures, accepted by Phys. Rev. D

Motivation Missingness in label-free mass spectrometry is inherent to the technology. A computational approach to recover missing values in metabolomics and proteomics datasets is important. Most existing methods are designed under a particular assumption, either missing at random or under the detection limit. If the missing pattern deviates from the assumption, it may lead to biased results. Hence, we investigate the missing patterns in free mass spectrometry data and develop an omnibus approach GMSimpute, to allow effective imputation accommodating different missing patterns. Results Three proteomics datasets and one metabolomics dataset indicate missing values could be a mixture of abundance-dependent and abundance-independent missingness. We assess the performance of GMSimpute using simulated data (with a wide range of 80 missing patterns) and metabolomics data from the Cancer Genome Atlas breast cancer and clear cell renal cell carcinoma studies. Using Pearson correlation and normalized root mean square errors between the true and imputed abundance, we compare its performance to K-nearest neighbors’ type approaches, Random Forest, GSimp, a model-based method implemented in DanteR and minimum values. The results indicate GMSimpute provides higher accuracy in imputation and exhibits stable performance across different missing patterns. In addition, GMSimpute is able to identify the features in downstream differential expression analysis with high accuracy when applied to the Cancer Genome Atlas datasets. Availability and implementation GMSimpute is on CRAN: https://cran.r-project.org/web/packages/GMSimpute/index.html. Supplementary information Supplementary data are available at Bioinformatics online.

Smart and versatile salicylaldehyde Schiff’s bases have been proved their excellent performances including large shocks shift, dual emission wavelengths and sensitive to environment for fluorescence analysis. Herein, a simple salicylaldehyde Schiff’s base molecular (PBAS) with aggregation-induced emission (AIE) and the excited-state intramolecular proton-transfer (ESIPT) effects was constructed for detecting N2H4 and ClO−. The highly specific and sensitive response to N2H4 was witnessed by the fast turn-on of the strong blue fluorescence and to ClO− was observed by the rapid turn off of the weak green fluorescence simultaneous decomposing of the probe. The results of mass spectrum analysis showed that probe PBAS decomposed under the influence of N2H4, whereas probe PBAS can complex with ClO− and prevent effective ESIPT process. Benefiting from its high properties, this fluorescence molecular provides an effective tool for probing N2H4 and ClO− in live cells.

In this work, we seriously discuss whether $X(3915)$ can be treated as a ${\ensuremath{\chi}}_{c0}(2P)$ state. Based on an unquenched quark model, we give the mass spectrum of the ${\ensuremath{\chi}}_{cJ}(2P)$ states, where there are no free input parameters in our calculation. Our result shows that the mass gap between ${\ensuremath{\chi}}_{c0}(2P)$ and ${\ensuremath{\chi}}_{c2}(2P)$ can reach 13 MeV, which can reproduce the mass difference between $Z(3930)$ and $X(3915)$. Additionally, the calculated masses of ${\ensuremath{\chi}}_{c0}(2P)$ and ${\ensuremath{\chi}}_{c2}(2P)$ are consistent with experimental values of $X(3915)$ and $Z(3930)$, respectively. Besides, giving the mass spectrum analysis to support $X(3915)$ as ${\ensuremath{\chi}}_{c0}(2P)$, we also calculate the width of ${\ensuremath{\chi}}_{c0}(2P)$ with the same framework, which is also consistent with the experimental data of $X(3915)$. Thus, the possibility of charmoniumlike state $X(3915)$ as ${\ensuremath{\chi}}_{c0}(2P)$ state is further enforced.

Dopant analysis in next-generation semiconductor devices has become increasingly difficult for traditionally used analytical techniques. Atom probe tomography has been viewed by some as a possible solution to these challenges because of its three-dimensional capabilities, forcing the atom probe to mature at a rapid pace in this particular field. This work presents a well-rounded analysis of how APT can be used to examine B dopant diffusion into the channel of a next-generation FinFET, where the channel dimensions and the number of dopants atoms are significantly smaller than any devices measured by APT to date. Complimentary EELS analysis of the gate and channel provides a better understanding of how distortions and artifacts in the APT reconstruction affect the overall integrity of the dataset. Dopant measurements in the channel are confirmed through in-depth mass spectrum analysis and compared with values proposed by TCAD modeling.

The objective of this study was to determine whether fish oil supplement has an effect on adipose tissue lipid profiles and gene expression in postpartum dairy cows. Holstein cows were supplemented with either long-chain n-3 polyunsaturated fatty acid (PUFA; protected fish oil) or control PUFA (n-6; toasted soybeans) for 2mo after calving (n=23 per diet). These cows showed no difference in milk production or metabolic parameters, but exhibited a tendency toward a decrease in early embryo mortality rate after artificial insemination. We hypothesized that, in addition to this effect, modifications in adipose tissue (AT) gene expression and lipid profiles would occur in response to diet. Subcutaneous AT samples were thus collected from the dewlaps of n-3 and n-6 dairy cows at 1mo antepartum, and 1wk, 2mo, and 5mo postpartum for the analysis of lipids and gene expression. Lipid profiles were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in both positive and negative modes. We found 37 lipid species in the 200 to 1,200 m/z range, which differed between the n-3 and control groups, suggesting that the n-3 supplement affected the lipid composition through the enrichment of lipids integrating long-chain PUFA from fish oil sources: eicosapentaenoic and docosahexaenoic acid. Moreover, a decrease in triacylglycerolipids was observed in AT of n-3 supplemented cows. The expression of 44 genes involved in fatty acid metabolism and the adipokine system was assessed by real-time reverse-transcription PCR. Hierarchical clustering, according to either postpartum stage or diet, enabled us to group genes exhibiting similar kinetic properties during lactation or by those that varied in similar ways after n-3 supplementation, respectively. Among the genes exhibiting a dietary effect, FABP4, LIPE, CD36, and PLIN1 were overexpressed in n-3 AT samples compared with the control, suggesting an increase in lipolysis due to n-3 supplementation, which was reflected on lipolytic activity at the protein level (i.e., protein expression of fatty acid binding protein 4, phosphorylated perilipin 1, and phosphorylated hormone-sensitive lipase). This increase in lipolysis is relevant to the decrease in triglycerides observed in these samples. Gene expression analyses between n-3 and control AT samples also suggested that the n-3 diet could modulate the secretory functions of AT, possibly by affecting adipokine expression; however, this has to be confirmed at the protein level.

Approaches enabling prediction of chocolate quality from cocoa composition would avoid time- and money-consuming steps to chocolate makers. Average mass spectra of cocoa-polyphenol-extracts led to fingerprints used to select the molecules that discriminate chocolate sensory groups.16 worldwide cocoa samples were processed into chocolates which were characterized by sensory analysis, allowing sorting of the samples into four sensory groups.The cocoa polyphenol extracts were analyzed by liquid chromatography−low-resolution mass spectrometry. Averaging each mass spectrum provided polyphenolic fingerprints, which were combined into a matrix and processed with chemometrics (PCA, PLS-DA) to select the most meaningful molecules for discrimination of the chocolate sensory groups.A larger set of 44 cocoa samples was used to validate the previous results. 29 mass signals of known and unknown molecules, mainly flavan-3-ols, were finally targeted[1], including 2 newly described ethyl-bridged flavan-3-ols[2], enabling sensory-group discrimination. Average mass spectra fingerprints of cocoa-polyphenol-extracts proved to be quick and efficient to select the molecules that discriminate chocolate sensory groups.A targeted MRM (Multiple Reaction Monitoring) mass spectrometry method was then developed and validated to routinely analyse large series of cocoa samples.[1] Fayeulle et al., Fast Discrimination of Chocolate Quality Based On Average Mass Spectra Fingerprints of Cocoa Polyphenols, J Agric. Food Chem., 2019, 67, 2723-2731[2] Fayeulle et al., Characterization of new flavan-3-ol derivatives in fermented cocoa beans, Food Chem., 2018 (259), 207-212

Part of special issue: The 5th International Conference on Food Digestion - Edited by Didier Dupont, Pasquale Ferranti, Alan Mackie 5. International Conference on Food Digestion (INFOGEST) ; Rennes (France) - (2017-04-04 - 2017-04-06) / Conférence; In the frame of the COST action INFOGEST, a static in vitro digestion protocol has been elaborated aiming at the improvement of data comparability by harmonizing the experimental conditions. The success in harmonization was confirmed with inter-laboratory trials using skim milk powder as a standardized model food. Moreover, the physiological relevance of the gastric and intestinal endpoints of the static digestion protocol was demonstrated in a pig in vivo trial, with the same skim milk powder and samples collected from different sections of the digestive tract, as well as in a human study with from jejunal effluents. In vivo, digestion is a dynamic process influenced by peristalsis and by the gradual secretion of enzymes and juices and the dwell time of the food. To mimic these physiological mechanisms, dynamic in vitro digestion protocols are widely used. Until now, the differences of protein hydrolysis taking place during dynamic and static in vitro digestion have not been investigated.In this study, the gradual hydrolysis of the main milk proteins present in skim milk powder was digested with the dynamic DIDGI®-system using adult digestion protocol and the static harmonized INFOGEST method. Protein hydrolysis was analyzed by gel electrophoresis, peptide patterns were measured with mass spectrometry, and free amino acids with high pressure liquid chromatography. The peptide patterns at the gastric and intestinal endpoints of in vitro digestion showed a good approximation to the in vivo results from pigs. Moreover, gradual peptide generation was comparable in both in vitro digestion conditions. However, the dynamic protocol reflected the physiological situation better at the level of free amino acid release. Nonetheless, in both in vitro digestion protocols, absorption of free amino acids is not simulated, and they are therefore limited in reflecting the in vivo situation at this level.

This work aims to sort cocoa beans according to chocolate sensory quality and phenolic composition. Prior to the study, cocoa samples were processed into chocolate in a standard manner, and then the chocolate was characterized by sensory analysis, allowing sorting of the samples into four sensory groups. Two objectives were set: first to use average mass spectra as quick cocoa-polyphenol-extract fingerprints and second to use those fingerprints and chemometrics to select the molecules that discriminate chocolate sensory groups. Sixteen cocoa polyphenol extracts were analyzed by liquid chromatography-low-resolution mass spectrometry. Averaging each mass spectrum provided polyphenolic fingerprints, which were combined into a matrix and processed with chemometrics to select the most meaningful molecules for discrimination of the chocolate sensory groups. Forty-four additional cocoa samples were used to validate the previous results. The fingerprinting method proved to be quick and efficient, and the chemometrics highlighted 29 m/ z signals of known and unknown molecules, mainly flavan-3-ols, enabling sensory-group discrimination.

Introduction The resistance of proteins to digestion may play a role in determining their allergenic potential. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriate when ethical constraints hinder in vivo studies. In our study, the ‘pepsin resistance test’, currently used by the European Food Safety Authority to assess allergenicity of proteins is compared to more physiologically relevant in vitro digestion protocols. A peptidomics workflow based on free softwares is used to identify, quantify and perform statistical analysis on the peptides produced. Methods Comparison of an infant, early phase adult, and late phase adult model is being applied on a panel of allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate in gastric and intestinal compartment is determined in order to detect persistent intact protein (SDS-PAGE) and hydrolysis products larger 9 amino acids (LC-MS). All peptidomics data (NanoLC fitted to Quadrupole-Orbitrap MS) were processed with free softwares: from data conversion to peptide identification, quantification and statistical analysis. Results More than two thousands NanoLC-MS/MS runs were acquired during the course of this project and the number of peptides identified ranged from one to five hundred per proteins depending on their resistance to enzymatic hydrolysis. All peptides identified (X!tandempipeline software [1]) were automatically quantified (MassChroQ [2]) in all samples, regardless the fact that it was identified at this particular time of digestion. Peptide quantification results processed in the R software environment allowed the clustering of peptides based on their behavior regarding the digestion protocol used and the digestion time (0,5 to 60min). Proteins that are not pepsin resistant show slower kinetics of proteolysis for the infant model of digestion, highlighting the fact that, for this population, they may still cause undesired immune responses via sensitization in the duodenum. Conclusions Differences in the kinetics of proteolysis have been found across models for proteins that are not pepsin resistant. This project highlights the importance of a multi-test protocol to assess protein digestibility. Novel Aspect Using a full pipeline of free softwares for peptidomics data treatment, we were able to point out peptide clusters which are only abundant when more physiologically relevant models of digestion were used.

The newly observed ${\mathrm{\ensuremath{\Sigma}}}_{b}(6097)$ provides us a good chance to further construct the high excited states of the bottom baryon family. In this work, we explain the ${\mathrm{\ensuremath{\Sigma}}}_{b}(6097)$ to be a $1P$ state with ${J}^{P}=5/{2}^{\ensuremath{-}}$ or $3/{2}^{\ensuremath{-}}$ by giving the mass spectrum analysis and the strong decay calculation, and predict the existence of $1P$ and $2S$ partners of the ${\mathrm{\ensuremath{\Sigma}}}_{b}(6097)$, where their masses and the corresponding decay behaviors are presented. Because of the success of describing the ${\mathrm{\ensuremath{\Sigma}}}_{b}(6097)$ and the former observed ${\mathrm{\ensuremath{\Xi}}}_{b}(6227)$ under the quasi-two-body treatment to the bottom baryons, we continue to predict the ${\mathrm{\ensuremath{\Omega}}}_{b}$ partners of ${\mathrm{\ensuremath{\Sigma}}}_{b}(6097)$ by the same method, which are the $1P$ and $2S$ states of the $bss$ baryon system. Identifying these predicted bottom baryons will be a research topic full of challenge and opportunity for future experiments, especially the LHCb.

Session 2: Microbial ecosystem and herbivore nutritionSession 2: Microbial ecosystem and herbivore nutrition; Changes in rumen metabolites from lambs fed a feed additive containing linseed oil and garlic essential oil. 10. International Symposium on the Nutrition of Herbivores (ISNH10)

Selecting the newly observed ${\mathrm{\ensuremath{\Xi}}}_{b}(6227{)}^{\ensuremath{-}}$ by LHCb as a study example, we decode its inner structure by giving the mass spectrum analysis and the investigation of its two-body strong decay behaviors. Our result indicates that the ${\mathrm{\ensuremath{\Xi}}}_{b}(6227{)}^{\ensuremath{-}}$ is a good candidate of the $P$-wave ${\mathrm{\ensuremath{\Xi}}}_{b}^{\ensuremath{'}}$ state with ${J}^{P}=3/{2}^{\ensuremath{-}}$ or $5/{2}^{\ensuremath{-}}$. In addition, we further provide the information of the properties of the partners of the ${\mathrm{\ensuremath{\Xi}}}_{b}(6227{)}^{\ensuremath{-}}$. These predicted states include three $2S$ states and the remaining $1P$ states in the bottom-strange baryon family. The calculated sizable Okubo-Zweig-Iizuka--allowed decay widths of these partners show that the experimental search for them becomes possible via LHCb. We have a reason to believe that the present study can be treated as a start point for constructing the highly excited bottom baryon spectroscopy.

The antibacterial activity of camel milk b-casein and its peptic hydrolysate was investigated. The hydrolysatewas fractionated by ultrafiltration successively through membranes with a 10 kDa and a 1 kDacut-off, sequentially. Antibacterial activity assays against Staphylococcus aureus CNRZ 3, Listeria innocuaATCC 33090 and Escherichia coli ATCC 25922 were performed. The growth of S. aureus and L. innocuastrain was not inhibited by b-casein; E. coli strain growth was slightly inhibited. All fractions of hydrolysatesexhibited some anti-bacterial activity and molecular mass fraction

An egg product is a complex matrix, mainly composed of water, proteins and lipids. It also contains carbohydrates, vitamins, minerals and many other compounds in varying concentrations. This chemical complexity makes it difficult to analyze, and most of the protocols are mainly concerned with the albumen and vitellus fractions, aside from the whole egg product. The hygienic quality and freshness of eggs can be assessed according to physico-chemical or microbiological criteria, imposed by the regulations in force. The state provides procedures and recommendations for the production and marketing of such products. Criteria and procedures vary from region to region, for example European legislation forbids the breaking of eggs by centrifugation for eggs intended for the food industry whereas American legislation allows the possibility of breaking eggs by centrifugation if they are clean, disinfected and intact and if they ultimately undergo heat treatment (baking etc.). In addition to the procedures and recommendations concerning the production process, the hygienic quality of egg products is ensured by controlling the physico-chemical and microbiological quality of the raw materials used and the finished products. European legislation thus provides maximum levels for certain compounds such as lactic acid or succinic acid at different points in the production chain.

Part of the Methods in Molecular Biology book series (MIMB, volume 1730); GC/MS-based metabolomics is a powerful tool for metabolic phenotyping and biomarker discovery from body biofluids. In this chapter, we describe an untargeted metabolomic approach for plasma/serum and fecal water sample profiling. It describes a multistep procedure, from sample preparation, oximation/silylation derivatization, and data acquisition using GC/QToF to data processing consisting in data extraction and identification of metabolites.

Stable-isotope labeling experiments (ILEs) are widely used to investigate the topology and operation of metabolic networks. The quality of isotopic data collected in ILEs is of utmost importance to ensure reliable biological interpretations, but current evaluation approaches are limited due to a lack of suitable reference material and relevant evaluation criteria. In this work, we present a complete methodology to evaluate mass spectrometry (MS) methods used for quantitative isotopic studies of metabolic systems. This methodology, based on a biological sample containing metabolites with controlled labeling patterns, exploits different quality metrics specific to isotopic analyses (accuracy and precision of isotopologue masses, abundances, and mass shifts and isotopic working range). We applied this methodology to evaluate a novel LC-MS method for the analysis of amino acids, which was tested on high resolution (Orbitrap operating in full scan mode) and low resolution (triple quadrupole operating in multiple reaction monitoring mode) mass spectrometers. Results show excellent accuracy and precision over a large working range and revealed matrix-specific as well as mode-specific characteristics. The proposed methodology can identify reliable (and unreliable) isotopic data in an easy and straightforward way and efficiently supports the identification of sources of systematic biases as well as of the main factors that influence the overall accuracy and precision of measurements. This approach is generic and can be used to validate isotopic analyses on different matrices, analytical platforms, labeled elements, or classes of metabolites. It is expected to strengthen the reliability of isotopic measurements and thereby the biological value of ILEs.

Phospholipids (PLs) represent a class of metabolites of interest for evaluating the relationship between diet and the development of several metabolic diseases. Given that PLs are rich in unsaturated fatty acids, they can be oxidized. Because of their structure and reactivity, oxidized phospholipids (PLs-Ox) are increasingly recognized as markers of oxidative stress and of various diseases associated with inflammation. Therefore, there is a growing interest in studying PLs-Ox in lipidomics. Because of their limited commercial availability, very little information is currently available in databases to identify these molecules. The aim of this study is to acquire new knowledge about PLs-Ox in order to propose an analytical strategy for their analyses. For this purpose, a synthesis method of PLs-Ox, in auto-oxidation, has been developed and applied on phosphatidylcholine and phosphatidylethanolamine molecular species with various chain lengths, degree, and position of unsaturations. An analysis method based on mass (MS) and tandem mass spectrometry coupled to electrospray ionization was then developed and enabled the identification of a great diversity of long- and short-chain oxidation products. Formation kinetics of oxidation products was evaluated. Results showed that the formation of oxidized compounds was largely influenced by the degree of unsaturation on fatty acid chains. Oxidation time promotes the formation of some biologically important oxidation products. Coupling the MS method with liquid chromatography in flow injection analysis mode enabled the development of a full analytical strategy. Structural analysis of PLs-Ox allowed the enrichment of databases with important information to identify these molecules in biological matrices.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine S'-triphosphate 3'-diphosphate (pppGpp) play a central role in the adaptation of bacterial and plant cells to nutritional and environmental stresses and in bacterial resistance to antibiotics. These compounds have historically been detected and quantified by two-dimensional thin-layer chromatography of P-32-radiolabeled nucleotides. We report a new method to quantify ppGpp and pppGpp in complex biochemical matrix using ion chromatography coupled to high-resolution mass spectrometry. The method is based on isotopic dilution mass spectrometry (IDMS) using C-13 to accurately quantify the nucleotides. However, the loss of a phosphate group from pppGpp during the sample preparation process results in the erroneous quantification of ppGpp. This bias was corrected by adding an extra N-15 isotope dilution dimension. This double-spike IDMS method was applied to quantify the ppGpp and pppGpp in Escherichia coli and in a mutant strain deleted for gppA (encoding the ppGpp phosphohydrolase) before and after exposure of both strains to serine hydroxamate, known to trigger the accumulation of these nucleotides.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi) catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-beta-glucosyl-(+)-catechin and 7-O-beta-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

This research has been supported by the ANR French program MetaboHUB. We would like to thank Mélanie Pétéra for her valuable technical assistance; AbstractIntroduction: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level.Objectives: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock.Methods: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA).Results: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function.Conclusions: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.

Background and Aims: Laccase enzymes produced by Botrytis cinerea are involved in the oxidation of phenolic substances during the development of grey mould, which causes significant economic losses. The aim of this work was to study the structural and activity characteristics of the laccase enzymes secreted by three B. cinerea strains that are involved in the development of grey mould. Methods and Results: Laccase enzymes obtained from three B. cinerea strains [one reference strain (B05.10) and two strains obtained from two French vineyards (VA612 and RM344)] were characterised. Analysis by LC-QTOF-MS revealed that the three strains contained a mixture of Laccase-2-BcLCC2 and Laccase-3-BcLCC7. The structural characteristics of the laccases from the three strains, such as molecular weight and glycosylation degree, were identical. Nevertheless, their catalytic activities were significantly different. Conclusions: Differences in catalytic activities could be due either to possible differences in the relative amount of Laccase-2- BcLCC2 and Laccase-3-BcLCC7 produced by each strain or to differences in the glycosidic fraction of the enzymes. Significance of the Study: The severity of the infection caused by B. cinerea may be not only related to the infection level but also to the strain involved.

Two series of compounds showing mass signals at m/z 605 and 893 (negative ionization mode) have been detected in fermented cocoa beans. This study objective is to identify these mass signals and characterize their structure in fermented cocoa samples. Our hypothesis is that these signals may correspond to ethyl-bridged flavan-3-ols resulting from flavan-3-ol condensation with acetaldehyde which is a microbial metabolite. Mass spectrometry was used to compare the retention times and mass fragmentation patterns between a model solution using epicatechin and procyanidin dimer B2, the major flavan-3-ols of cocoa, as precursors and extracts of fermented cocoa. Their identification was confirmed: four isomers of ethyl-linked epicatechin as well as several isomers of epicatechin-ethyl-procyanidin B2, in which B2 was mostly linked through its upper unit, were characterized in cocoa. This study demonstrates the presence of flavan-3-ol acetaldehyde condensation products in fermented cocoa beans and provides the first report of epicatechin-ethyl-procyanidin B2.

Bacillus thuringiensis has been widely used as a bio-insecticide. However, novel biological activities other than insect toxicity of B. thuringiensis are still underestimated. In this study, a new lipopeptide biosynthesis gene cluster in B. thuringiensis BMB171 was discovered by genome mining and verified by reverse genetics. Thumolycin, the lipopeptide synthesized by this gene cluster, was then isolated and purified. Mass spectrum analysis revealed the molecular mass of thumolycin is 696.51 Da with the predicted molecular formula of C38H64N8O4. Further bioactivities assay showed that thumolycin endowed B. thuringiensis BMB171 with broad spectrum antimicrobial and nematocidal activities.

We thank Yunfei Liu for his contribution concerning correlation network analyses. All metabolomics analyses were performed within the metaboHUB French infrastructure (ANR-INBS-0010). This study was supported by the INRA DID'IT Metaprogramme. The GAZEL cohort received approvals from the National Commission for Data Processing and Freedoms (CNIL), the National Medical Council and the National Consultative Committee of Ethics, and the INSERM IRB. The study protocol was approved by the GAZEL Scientific Committee. All volunteers gave written and informed consent for this study. All the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report; The evolution of human health is a continuum of transitions, involving multifaceted processes at multiple levels, and there is an urgent need for integrative biomarkers that can characterize and predict progression toward disease development. The objective of this work was to perform a systems metabolomics approach to predict metabolic syndrome (MetS) development. A case-control design was used within the French occupational GAZEL cohort (n =112 males: discovery study n = 94: replication/validation study). Our integrative strategy was to combine untargeted metabolomics with clinical, sociodemographic, and food habit parameters to describe early phenotypes and build multidimensional predictive models. Different models were built from the discriminant variables, and prediction performances were optimized either when reducing the number of metabolites used or when keeping the associated signature. We illustrated that a selected reduced metabolic profile was able to reveal subtle phenotypic differences 5 years before MetS occurrence. Moreover, resulting metabolomic markers, when combined with clinical characteristics, allowed improving the disease development prediction. The validation study showed that this predictive performance was specific to the MetS component. This work also demonstrates the interest of such an approach to discover subphenotypes that will need further characterization to be able to shift to molecular reclassification and targeting of MetS

Development of a Mass Spectrometry Imaging (MSI) approach to explore Listeria monocytogenes biofilms exposed to a dehumidification stress. 8. Colloque du Réseau National Biofilms

Remerciements : Institut National du Cancer (INCa) / Institut Fédératif de Recherche biomédicale (IFRB) / Canceropôle Ilede- France / Réseau NACReRemerciements : Institut National du Cancer (INCa) / Institut Fédératif de Recherche biomédicale (IFRB) / Canceropôle Ilede-France / Réseau NACRe; Introduction et but de l’étude : La métabolomique permet d’étudier l’ensemble des métabolites présents dans unbiofluide. Cette étude basée sur une analyse non ciblée en spectrométrie de masse a pour but d’étudier si des profils métabolomiques établis à partir d’un prélèvement sanguin sur des femmes à priori en bonne santé, pourrait contribuer à prédire le risque à long terme de développer le cancer du sein et d’améliorer la compréhension de son étiologie.Matériel et méthodes : Un cas-témoin niché prospectif a été mené dans la cohorte SU.VI.MAX, incluant 211 cas de cancer du sein et 211 témoins appariés. Les profils métabolomiques ont été établis sur des échantillons de plasma prélevés à l’inclusion, avant l’apparition du cancer, grâce à une analyse non ciblée par spectrométrie de masse (LC-MS). Des modèles de régressions logistiques conditionnelles multivariées ont été calculés sur chaque ion présélectionné grâce à une ANOVA et sur les combinaisons de potentiels biomarqueurs issus d’une Analyse en Composante Principale. Une correction de test multiple de type FDR (False Discovery Rate) a été appliquée.Résultats et Analyse statistique : Plusieurs métabolites étaient associés au risque de cancer du sein. La finalisation de l’identification des métabolites discriminants est en cours. Au vu des résultats actuels, les femmes caractérisées par un niveau plasmatique plus élevé de valine (OR=1,45 [1,15-1,83]; p= 0.009), de phénylalanine (OR=1,43 [1,14-1,78]; p=0,009), de tryptophane (OR=1,4 [1,1-1,79]; p=0,01), et de glutamine (OR=1,33 [1,07-1,66]; p=0,01) et d’un niveau plus bas d’OSuccinyl-L-homoserine (OR=0,7 [0,55-0,89]; p=0,01) auraient un risque plus important de développer le cancer du sein dans la décennie qui suit.Conclusion : Cette étude suggère plusieurs associations entre profils métabolomiques à l’inclusion et risque à long terme de développer le cancer du sein et confirme certains résultats que nous avons mis en évidence par une analyse RMN. Cette étude pourrait aider à améliorer l’identification des femmes à risque de développer un cancer du sein, bien avant l’apparition des symptômes. L’étude des biomarqueurs nutritionnels associés au risque de développer cette pathologie (actuellement en cours) pourra permettre de préciser le rôle de la nutrition (facteur modifiable) dans l’étiologie du cancer du sein et d’améliorer les stratégies préventives.

N-acryloyl rhodamine B hydrazide, a non-responsive control of a colorimetric Cu(2+) sensor, was used as a turn-on fluorescent sensor for Hg(II) from HgCl2 in the presence of AgNO3. The detection was highly selective and sensitive, and a large number of environmentally and biologically relevant metal ions, such as Na(+), K(+), Ca(2+), Mg(2+), Fe(3+), Cu(2+), Zn(2+), Cr(3+), Pb(2+), Ni(2+), Fe(2+), Mn(2+), Co(2+), Cd(2+), including Hg(II) from the easy dissociated salts, did not show significant interference. The fluorescence of the sensor (10 μM) was enhanced 74 folds by 10 equiv. of Hg(II) from HgCl2 in THF/HAc-NaAc (1/1, v/v, pH = 6) aqueous buffer solution containing 20 equiv. of AgNO3. The maximal fluorescence intensity increased linearly with the concentration of Hg(II) in the range of 0-70 μM. The detection limit of Hg(II) was 0.59 μM. The sensing mechanism was explored by Job's plot experiment, reversible experiment, mass spectrum analysis, spectroscopic analysis, and thin-layer chromatography.

Hydroxylated polyhalodiphenyl ethers (HO-PXDEs) are emerging aquatic pollutants. Previous studies have shown that HO-PXDEs can photogenerate dioxins and phenolic compounds. However, it is unclear which photochemical pathways are responsible for the various photoproducts. This study investigates the direct photolysis and photooxidation initiated by (1)O2 and ˙OH that can be formed by photosensitization, taking 2'-HO-2,3',4,5'-tetrabromodiphenyl ether (2'-HO-BDE-68) as a case study. The results show that 1,3,8-tribromodibenzo-p-dioxin can only be produced during direct photolysis. By mass spectrum analysis, four dihydroxylated polybromodiphenyl ethers, generated from both direct and indirect photodegradation were confirmed. Among them, di-HO-tribromodiphenyl ether (di-HO-TBDE) was the main product generated from direct photohydrolysis. Most probably, the di-HO-TBDE is 2',5'-HO-2,3',4-tribromodiphenyl ether, as was suggested by density functional theory calculations. Ether bond cleavage is a dominant pathway for the direct photolysis and photooxidation reactions leading to 2,4-dibromophenol as the dominant product. The yields of the products, which are irrespective of reaction time and can be employed to compare the ability of different HO-PXDEs to photogenerate a given product, were reported. This study indicates that for accurate ecological risk assessment of HO-PXDEs, their different photodegradation pathways that may lead to different photoproducts should be considered.

MALDI imaging and profiling mass spectrometry approach for the analysis of Listeria monocytogenes biofilms exposed to a desiccation. SMMAP 2017, Spectrométrie de Masse, Métabolomique et Analyse Protéomique

Comparison of lipid profiles and gene expression in granulosa and cumulus cells in bovine. 33. Annual Meeting of the European Embryo Transfer Association (AETE)

Development of a MALDI imaging mass spectrometry (IMS) approach to bacterial proteomics: first application to Listeria monocytogenes biofilms exposed to a desiccation. 7. Congress of European Microbiologists FEMS 2017

In oviparous, the embryonic development depends on the extra-embryonic structures, namely the yolk, amnioticand allantoic sacs. To better appreciate their role in the development of the chicken embryo, we analyzed thephysicochemical properties and biological activities of the amniotic (LA) and allantoic (LC) fluids from the 8thto 16th day of incubation. We showed that LA pH remains stable throughout the incubation (~7.5), while LC pHand osmolality values decrease significantly. Furthermore, from day 12 onwards, drastic changes in LA proteinpattern have been observed by SDS-PAGE analysis, with the transfer of egg white proteins in the amniotic sac,whereas the LC protein composition remains stable with proteins mainly coming from the egg yolk. Moreover,the LC major proteins identified by mass spectrometry are associated with lipid and vitamin metabolisms, aswell as in metal ion transport, while the LA major proteins refer to embryo’s nutrition and protection againstpathogens. On the other hand, active proteases have been identified in the two fluids using gelatin zymography.By mass spectrometry analysis, a total of 12 proteases was detected in the LC, compared to only 5 in the LA.These proteases are defined to be involved in morphogenesis and tissue remodeling, and 4 of them, uniquelyfound in the LC, are digestive proteases. Altogether, these data give new insights in the respective properties androles of LA and LC in embryo development. This study will allow us to identify biomarkers reflecting a goodembryonic development, to evaluate the impact of in ovo injections (vaccines, probiotics) on LA and LCcompositions and explore their repercussions on the development of the embryo and chick health., Chez les ovipares, le développement de l’embryon repose sur des structures extra-embryonnaires telles que les sacs vitellin, amniotique et allantoïque. Pour comprendre l’implication de ces structures dans le développement de l’embryon chez la poule, nous avons caractérisé les propriétés physicochimiques et les activités biologiques des fluides allantoïque (LC) et amniotique (LA) du 8ème au 16ème jour de l’incubation. Nous avons montré que le pH du LA reste stable tout au long de l’incubation (~7,5) tandis que le pH et l’osmolalité du LC diminuent significativement. En parallèle, des changements drastiques dans la composition du LA ont été observés par SDS-PAGE à partir du 12ème jour, avec le transfert des protéines du blanc dans le sac amniotique, alors que le LC conserve une composition protéique stable, constituée majoritairement de protéines du jaune. Une étude complémentaire par spectrométrie de masse a permis de définir les protéines majeures du LC comme impliquées dans le métabolisme des vitamines et lipides, ainsi que dans le transport d’ions métalliques, alors que celles du LA sont des protéines vraisemblablement impliquées dans la nutrition de l’embryon et dans sa protection contre les pathogènes. D’autre part, des activités protéolytiques ont été visualisées par zymographie et une analyse par spectrométrie de masse a permis d’identifier 12 protéases dans le LC contre seulement 5 dans le LA. Ces protéases sont décrites comme impliquées dans la morphogénèse et la régénération de tissus, et 4 d’entre-elles, spécifiquement identifiées dans le LC, sont des protéases digestives. Ces résultats suggèrent une implication importante des deux fluides dans les processus du développement embryonnaire. A terme, cette étude permettra d’identifier les paramètres et les molécules témoignant du bon développement de l’embryon, et d’évaluer l’impact d’injections in ovo dans ces fluides (vaccins/probiotiques) sur leur composition, et leur éventuelle répercussion sur le développement de l’embryon et la santé du futur poussin.

Haemophilus influenzae is associated with severe invasive disease, while Haemophilus haemolyticus is considered part of the commensal flora in the human respiratory tract. Although the addition of a custom mass spectrum library into the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system could improve identification of these two species, the establishment of such a custom database is technically complicated and requires a large amount of resources, which most clinical laboratories cannot afford. In this study, we developed a mass spectrum analysis model with 7 mass peak biomarkers for the identification of H. influenzae and H. haemolyticus using the ClinProTools software. We evaluated the diagnostic performance of this model using 408 H. influenzae and H. haemolyticus isolates from clinical respiratory specimens from 363 hospitalized patients and compared the identification results with those obtained with the Bruker IVD MALDI Biotyper. The IVD MALDI Biotyper identified only 86.9% of H. influenzae (311/358) and 98.0% of H. haemolyticus (49/50) clinical isolates to the species level. In comparison, the ClinProTools mass spectrum model could identify 100% of H. influenzae (358/358) and H. haemolyticus (50/50) clinical strains to the species level and significantly improved the species identification rate (McNemar's test, P < 0.0001). In conclusion, the use of ClinProTools demonstrated an alternative way for users lacking special expertise in mass spectrometry to handle closely related bacterial species when the proprietary spectrum library failed. This approach should be useful for the differentiation of other closely related bacterial species.

International audience; The sample matrix can enhance the gas chromatography signal of pesticide residues relative to that obtained with the same concentration of pesticide in solvent. This paper is related to negative matrix effects observed in coupled gas chromatography-mass spectrometry ion trap (GC/MS2) quantification of pesticides in concentrated extracts of apple peel prepared by the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method. It is focused on the pesticides most frequently used on the apple varieties studied, throughout the crop cycle, right up to harvest, to combat pests and diseases and to improve fruit storage properties. Extracts from the fleshy receptacle (flesh), the epiderm (peel) and fruit of three apple varieties were studied by high-performance thin-layer chromatography hyphenated with UV–vis light detection (HPTLC/UV visible). The peel extracts had high concentrations of triterpenic acids (oleanolic and ursolic acids), reaching 25mgkg−1, whereas these compounds were not detected in the flesh extracts (

Remerciements :INRA, UMR PRC 0085, Plateforme CIRE, 37380 NouzillyStephane Fabre and Alan McNeilly for helpful discussionYves Combarnous for helpful suggestions, Maxime Capelle and Marie Champion for technical assistanceElodie Chaillou for critical reading of the manuscript; Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance (SPR) and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). SPR and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C (VWC) domain of TSP-1 is likely responsible for this BMP-2/4 binding activity, an assertion based on sequence similarity that TSP-1 shares with the VWC domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via the blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Grape pomace (GP) is known to be a rich source of polyphenols with biological activity which may be used as functional ingredients for the development of new health-promoting products. Numerous studies have reported that bioactive compounds may act through multiple mechanisms. In order to verify the oral absorption and metabolism of grape polyphenols, we performed a prospective, randomised and cross-over acute study in 12 volunteers with two interventions: 500 mL of a functional beverage enriched with 200 mL of GP and 500 mL of a control beverage without GP. In this work, liquid chromatography coupled with an electrospray ionization hybrid linear ion trap quadrupole-Orbitrap-mass spectrometry (LC/ESI-LTQ-Orbitrap-MS) technique has been used to accurately identify phenolics in GP and human urine. In GP, 41 phenolic compounds were identified mainly procyanidins, phenolic acids and flavonols, and in human urine over 70 metabolites of phenolic compounds including microbiota metabolites, glucuronides and sulfate derivatives were detected. Overall, high resolution mass spectrometry (HR-MS) enhances the identification of a large variety of polyphenols and their metabolites with great mass accuracies for all molecular ions.

The authors are grateful to Dr David Touboul, Isabelle-Schmitz-Afonso (ICSN-CNRS), Dr Marie Mejean and Marine Leblanc for fruitful discussions and guidance, and to Estella Vallade for technical assistance. Natacha Bonneau is the recipient of a doctoral grant from the Ministere de l'enseignement superieur et de la recherche (France); Annonaceous acetogenins (AAGs) are neurotoxins possibly responsible for atypical Parkinsonism/dementiaclusters, via the consumption of edible Annonaceae fruits. Their presence was investigated in fruitpulps of Annona squamosa from different locations. Qualitative analysis of other AAGs was performed.We here report the identification of squamocin in batches from Asia, the Caribbean Basin and theIndian Ocean. This molecule was quantified by HPLC-UV, evidencing a content of 13.5–36.4 mg/fruit.HPLC-ESI-Q-TOF allowed the detection of 25 different raw formulas matching with AAGs. LC–MS/MSmethodological development was performed using 4 representative standards. The main AAGs couldbe annotated, including bullatacin (rolliniastatin-2) and annonacin. This study evidences a remarkablehomogeneity for the main AAGs within the species, and discrepancies for minor compounds. These findingsindicate that A. squamosa should be considered a risk factor for neurodegenerative disorders

Mozzarella cheese is a classical dairy product but most research to date has focused on low moisture products. In this study, the microstructure and physicochemical properties of both laboratory and commercially produced high moisture buffalo Mozzarella cheeses were investigated and compared to high moisture bovine products. Buffalo and bovine Mozzarella cheeses were found to significantly differ in their microstructure, chemical composition, organic acid and proteolytic profiles but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat to protein and a microstructure containing larger fat patches and a less dense protein network. Liquid chromatography mass spectrometry detected the presence of only β-casein variant A2 and a single β-lactoglobulin variant in buffalo products compared to the presence of both β-casein variants A1 and A2 and β-lactoglobulin variants A and B in bovine cheese. These differences arise from the different milk composition and processing conditions. The differences in microstructure and physicochemical properties observed here offer a new approach to identify the sources of milk used in commercial cheese products.

Cost-effective and environment-friendly pretreatments, such as fungal pretreatments, are required for anaerobic digestion of lignocellulosic biomass particularly because the amount of methane produced is often limited by the lignin content. Anaerobic digestibility is estimated using a Biochemical Methane Potential (BMP) test, which lasts several weeks. Since the Py-GC–MS technique is considered to be a rapid method for obtaining information on various organic components, its suitability for the study of fungal pretreatment efficiency in anaerobic digestion was investigated here, to our knowledge for the first time. In this goal, mycelium of the white-rot fungi Polyporus brumalis BRFM 985, untreated wheat straw and straw pretreated with different fungal strains and under different conditions were analysed with Py-GC–MS. In the mycelium pyrolysate, diverse compounds, often considered as unspecific, probably derive from proteins (toluene, etc.). A strong presence of tyrosine and phenylalanine among the fungus amino acids was also suspected. As for pretreated straw samples, a correlation was observed between the amount of fungal biomass determined by qPCR (used as a reference method) and the sum of relative areas of toluene, styrene and ethylbenzene in the pyrograms, showing that it is feasible to estimate the fungal biomass amount on pretreated straws using Py-GC–MS. In addition, the H/L-Py (Holocelluloses/Lignin) ratio, determined by dividing the sums of areas of pyrolysis compounds that have a polysaccharide (PS) and lignin (LIG) origin, was correlated to the BMP values of pretreated straws, thus showing that the pretreatment efficiency can be rapidly estimated with Py-GC–MS in the tested conditions.

International audience; Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.

A UHPLC-MS/MS method was developed for the quantification of the main compounds involved in oxidation reactions occurring in white musts and wines such as hydroxycinnamic acids, their glutathione and cysteinylglycine adducts (GRP, GRP2, 5-(S-glutathionyl)-trans-caftaric acid, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid), and reduced and oxidized glutathione (GSH, GSSG) in wine. Since oxidation is the main concern in white wine-making, directly affecting its quality, the developed method was then applied in a series of white wines made with different pre-fermentation treatments to limit oxidation at must stage. The glucose esters and/or glucosides of hydroxycinnamic acids were quantified as glucogallin equivalent. The developed method led to an overall improvement in the limits of detection (LODs) and quantification (LOQs) for all the compounds studied in comparison to other methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD) or diode array UV detection (HPLC-DAD). LOD values ranged from 0.0002 to 0.0140 mg/L and LOQs from 0.0005 to 0.0470 mg/L. The recoveries ranged between 80 and 110% in wines, and the relative standard deviation (RSD) for precision intra- and inter-day was below 15%. The accuracy and intra- and inter-day precision met the acceptance criteria of the AOAC international norms. As far as we know, this study is the first report of quantification of GRP, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid using these non-commercially available compounds as external standards. Those compounds represent a significant proportion of hydroxycinnamic acid derivatives in wines. The methodology described is suitable for the analysis of hydroxycinnamic derivatives in wines.

In amniotes, the amniotic fluid is a significant contributor to fetal development and health. While numerous studies have been conducted in mammalian amniotic fluid, the composition of amniotic and other extraembryonic fluids in avian egg along with their physiological functions remain largely unexplored. In such a context, our objective was to characterize the chicken amniotic fluid (AmF) and allantoic fluid (AlF) properties, protein composition, and some associated functions from day 8 to day 16 of incubation. SDS-PAGE combined to mass spectrometry analysis revealed common and specific proteins to each fluid, suggesting distinct properties and functions. Indeed, major AlF proteins are mostly “egg yolk” proteins involved in lipid, vitamin metabolisms, and metal ion transport, while major AmF proteins resemble those of albumen. Drastic changes in the AmF protein profiles were observed during incubation, when the albumen transfers from day 12 onwards, while few changes were detected for the AlF protein profile. The decreases in osmolality (from 231 to 183 mOsm/kg) and pH (from 8.26 to 7.26) observed in the AlF during incubation are associated with water and electrolytes reallocation for the embryo needs. In contrast, AmF pH value remained stable (≈7.5). Active proteolytic enzymes have been identified in the 2 fluids using gelatin zymography, followed by mass spectrometry analysis for protease identification. A total of 12 proteases was detected in the AlF, compared to 5 in the AmF. We have shown that AlF concentrates proteolytic enzymes assumed to participate in digestive processes: aminopeptidase N, dipeptidyl peptidase-4, meprin A, and 72 kDa type IV collagenase preproprotein. The other proteases identified in both fluids also could have a role in morphogenesis (hepatocyte growth factor activator, suppressor of tumorigenicity 14, astacin-like metalloendopeptidase) and hemostasis (prothrombin and coagulation factor X). Altogether, these data suggest that the roles of chicken AlF and AmF are not merely associated with protection of the embryo and regulation of metabolic disposable wastes, but also they could have more sophisticated roles during embryonic development.