A sportomics soccer investigation unveils an exercise-induced shift in tyrosine metabolism leading to hawkinsinuria.

Tyrosine metabolism has an intense role in the synthesis of neurotransmitters. Our study used an untargeted, sportomics-based analysis of urine samples to investigate changes in metabolism during a soccer match in 30 male junior professional soccer players. Samples were collected before and after the match and analyzed using liquid chromatography and mass spectrometry. Results showed significant changes in tyrosine metabolism. Exercise caused a downregulation of the homogentisate metabolites 4-maleylacetoacetate and succinylacetone to 20% (p = 4.69E-5) and 16% (p = 4.25E-14), respectively. 4-Hydroxyphenylpyruvate, a homogentisate precursor, was found to be upregulated by 26% (p = 7.20E-3). The concentration of hawkinsin and its metabolite 4-hydroxycyclohexyl acetate increased ~six-fold (p = 1.49E-6 and p = 9.81E-6, respectively). Different DOPA metabolism pathways were also affected by exercise. DOPA and dopaquinone increased four-to six-fold (p = 5.62E-14 and p = 4.98E-13, respectively). 3-Methoxytyrosine, indole-5,6-quinone, and melanin were downregulated from 1 to 25%, as were dopamine and tyramine (decreasing to up to 5% or 80%; p= 5.62E-14 and p = 2.47E-2, respectively). Blood TCO2 decreased as well as urinary glutathione and glutamate (40% and 10% respectively) associated with a two-fold increase in pyroglutamate. Our study found unexpected similarities between exercise-induced changes in metabolism and the inherited disorder Hawkinsinuria, suggesting a possible transient condition called exercise-induced hawkinsinuria (EIh). Additionally, our research suggests changes in DOPA pathways may be involved. Our findings suggest that soccer exercise could be used as a model to search for potential countermeasures in Hawkinsinuria and other tyrosine metabolism disorders.

Plasma proteomic signature of major depressive episode in the elderly.

Depression is a complex and multifactorial disease, affecting about 6.5% of the elderly population in what is referred to as late-life depression (LLD). Despite its public health relevance, there is still limited information about the molecular mechanisms of LLD. We analyzed the blood plasma of 50 older adults, 19 with LLD and 31 controls, through untargeted mass spectrometry, and used systems biology tools to identify biochemical pathways and biological processes dysregulated in the disease. We found 96 differentially expressed proteins between LLD patients and control individuals. Using elastic-net regression, we generated a panel of 75 proteins that comprises a potential model for determining the molecular signature of LLD. We also showed that biological pathways related to vesicle-mediated transport and voltage-dependent calcium channels may be dysregulated in LLD. These data can help to build an understanding of the molecular basis of LLD, offering an integrated view of the biomolecular alterations that occur in this disorder. SIGNIFICANCE: Major depressive disorder in the elderly, called late-life depression (LLD), is a common and disabling disorder, with recent prevalence estimates of 6.5% in the general population. Despite the public health relevance, there is still limited information about the molecular mechanisms of LLD. The findings in this paper shed light on LLD heterogeneous biological mechanisms. We uncovered a potential novel biomolecular signature for LLD and biological pathways related to this condition which can be targets for the development of novel interventions for prevention, early diagnosis, and treatment of LLD.

Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics.

Proteomic tools are especially useful when it comes to investigating complex samples such as human blood plasma, in which protein quantities can span across up to ten orders of magnitude. Ultra definition mass spectrometry, in combination with two-dimensional liquid chromatography, provides better coverage of complex proteomes and allows for better control of collision energy, keeping the fragmentation benefits of high collision energy associated with drift time measurements from ion mobility separation. Here, we present a protocol to assist in the identification of proteins in human blood plasma and other similar samples with a large dynamic range.

Tri- and dipeptides identification in whey protein and porcine liver protein hydrolysates by fast LC-MS/MS neutral loss screening and de novo sequencing.

We describe a fast (5 min) liquid chromatography tandem mass spectrometry method (LC-MS/MS) based on a 46 Da neutral loss of formic acid (H2 O and CO) to identify tri- and dipeptides (DIPEP) in whey protein and porcine liver protein hydrolysates and confirmed by further de novo sequencing. Sample solutions were acidified to favor [dipep + H]+ ions, and a m/z range of 50-300 was used to improve sensitivity. All dipeptide candidates were selected based on all possibilities of the 20 amino acid combinations, and their collision-induced dissociation fragments were screened via de novo sequencing. To determine their biological activities, sequenced dipeptides were compared with the Biopep database and other data from literature. Altogether, 18 dipeptides and 7 tripeptides were identified from the whey protein hydrolysate; they seemed to be broadly active, and peptides were identified as active dipeptidyl peptidase IV inhibitors and active angiotensin-converting enzyme (ACE), according to available information. Porcine liver hydrolysate showed 14 dipeptides which exhibit similar biological activities to whey protein hydrolysate.

Proteome alterations associated with the oleic acid and cis-9, trans-11 conjugated linoleic acid content in bovine skeletal muscle.

Oleic acid (OA) and cis-9, trans-11 conjugated linoleic acid (c9t11-CLA) are fatty acids found in beef with beneficial effects in human health. This study investigated differentially abundant proteins (DAPs) in skeletal muscle of bovines with extreme values of OA, and c9t11-CLA. For each one of the fatty acids, twenty muscle samples were divided into two groups (N = 10_High; N = 10_Low) and analyzed by high definition mass spectrometry. We identified 103 and 133 DAPs between the groups for each fatty acid. We found 64 and 45 up-regulated and 39 and 68 down-regulated proteins for OA and c9t11-CLA, respectively. Comparative analysis between proteomic and transcriptomic data revealed eight and ten genes with a consistent between mRNA expression levels and protein abundance for OA and c9t11-CLA, respectively. Unconventional myosin-Id (MYO1D), mineralocorticoid receptor (NR3C2), geranylgeranyl transferase type-2 subunit-alpha (RABGGTA), and uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA) were found as putative candidate proteins for OA content. Fatty acid synthase (FASN), tubulin alpha-4A chain (TUBA4A), vinculin (VCL), NADH dehydrogenase 1 alpha subcomplex 5 (NDUFA5), and prefoldin subunit 6 (PFDN6) for c9t11-CLA. Our findings contribute to a deeper understanding of the molecular mechanisms behind the regulation of the OA and c9t11-CLA content in cattle skeletal muscle. SIGNIFICANCE: Questions about the association between meat intake and disease incidence in humans has driven animal scientist to pursue a better understanding of the biological processes associated with differences in the intramuscular fat composition. The beneficial effects of oleic acid and conjugated linoleic acid in human health have been demonstrated by improving the immune system and preventing atherosclerosis, different types of cancers, hypertension, and diabetes. Previous genome-wide association and gene expression studies identified genomic regions and differentially expressed genes associated with the fatty acid profile in skeletal muscle. In this work, differences were evaluated at the protein level. The use of a label-free quantitative proteomic approach, compared with muscle transcriptome results obtained by RNA-sequencing, allowed us to earn new insights into the variability in fatty acid deposition in skeletal muscle of farm animals. This study opens new avenues to explore the effect of the fatty acids in the skeletal muscle of livestock animals, which is associated with nutritional values of the meat, and perhaps to understand the mechanisms correlated with metabolic diseases in other species.

Saliva proteomics from children with caries at different severity stages.

OBJECTIVE: To perform a comparative analysis of saliva protein profile of patients with early childhood caries at different levels of severity and caries-free individuals. MATERIALS AND METHODS: Stimulated saliva samples were collected from 126 children (2-6 years old), classified according to the ICDAS II, and divided into 3 groups (n = 42): caries-free (CF), enamel caries (EC), and dentine caries (DC). Samples were digested and analyzed by nanoUPLC coupled with a mass spectrometry. Data analyses were conducted with Progenesis QI for Proteomics Software v2.0. Gene Ontology (GO) terms and protein-protein interaction analysis were obtained. RESULTS: A total of 306 proteins (≈6 peptides) were identified. Among them, 122 were differentially expressed in comparisons among children with different caries status. Out of the 122 proteins, the proteins E2AK4 and SH3L2 were exclusively present in groups CF and EC, respectively, and 8 proteins (HAUS4, CAH1, IL36A, IL36G, AIMP1, KLHL8, KLH13, and SAA1) were considered caries-related proteins when compared to caries-free children; they were up-regulated proteins in the caries groups (EC and DC). CONCLUSION: The identification of exclusive proteins for caries-free or carious-related conditions may help in understanding the mechanisms of caries and predicting risk as well as advancing in caries control or anti-caries approaches.

Immune Response Resetting in Ongoing Sepsis.

Cure of severe infections, sepsis, and septic shock with antimicrobial drugs is a challenge because morbidity and mortality in these conditions are essentially caused by improper immune response. We have tested the hypothesis that repeated reactivation of established memory to pathogens may reset unfavorable immune responses. We have chosen for this purpose a highly stringent mouse model of polymicrobial sepsis by cecum ligation and puncture. Five weeks after priming with a diverse Ag pool, high-grade sepsis was induced in C57BL/6j mice that was lethal in 24 h if left untreated. Antimicrobial drug (imipenem) alone rescued 9.7% of the animals from death, but >5-fold higher cure rate could be achieved by combining imipenem and two rechallenges with the Ag pool (p < 0.0001). Antigenic stimulation fine-tuned the immune response in sepsis by contracting the total CD3+ T cell compartment in the spleen and disengaging the hyperactivation state in the memory T subsets, most notably CD8+ T cells, while preserving the recovery of naive subsets. Quantitative proteomics/lipidomics analyses revealed that the combined treatment reverted the molecular signature of sepsis for cytokine storm, and deregulated inflammatory reaction and proapoptotic environment, as well as the lysophosphatidylcholine/phosphatidylcholine ratio. Our results showed the feasibility of resetting uncontrolled hyperinflammatory reactions into ordered hypoinflammatory responses by memory reactivation, thereby reducing morbidity and mortality in antibiotic-treated sepsis. This beneficial effect was not dependent on the generation of a pathogen-driven immune response itself but rather on the reactivation of memory to a diverse Ag pool that modulates the ongoing response.

Investigating the Potential of Ion Mobility-Mass Spectrometry for Microalgae Biomass Characterization.

Algae biomass is formed by an extremely complex set of metabolites, and its molecular characterization has been very challenging. We report the characterization of microalgae extracts via traveling wave ion mobility-mass spectrometry (TWIM-MS) by two different analysis strategies. First, the extracts were analyzed by direct infusion electrospray ionization (ESI) with no previous chromatographic separation (DI-ESI-TWIM-MS). Second, the samples were screened for metabolites and lipids using an untargeted high-throughput method that employs ultrahigh-performance liquid chromatography (UHPLC) using data-independent analysis (DIA) - MSE (UHPLC-HDMSE). Sixteen different microalgae biomasses were evaluated by both strategies. DI-ESI-TWIM-MS was able, via distinct drift times, to set apart different classes of metabolites, with the differences in the profiles of each microalga readily evident. With the UHPLC-HDMSE approach, 1251 different compounds were putatively annotated across 16 samples with 210 classified as lipids. From the normalized abundance for each annotated compound category, a detailed profiling in terms of metabolites, lipids, and lipid classes of each sample was performed. The reported workflow represents a powerful tool to determine the most suitable biotechnological applications for a given alga type and may allow for real-time monitoring of the algae composition distribution as a function of growth conditions, feedstocks, and the like. The determination of collision cross section results in improved confidence in the identification of triacylglycerols in samples, highly applicable to biofuels production. The two analysis strategies explored in this work offer powerful tools for the biomass industry by aiding in the identification of ideal strains and culture conditions for a specific application, saving analysis time and facilitating identification of a large number of constituents at once.

Data from proteomic analysis of bovine Longissimus dorsi muscle associated with intramuscular fat content.

The proteomic data presented in this article are associated with the research article entitled "Longissimus dorsi muscle label-free quantitative proteomic reveals biological mechanisms associated with intramuscular fat deposition" published in Journal of Proteomics [1]. In this article, we characterized the proteomic profile of bovine Longissimus dorsi muscle from Nelore steers and identified differentially abundant proteins associated with the intramuscular fat (IMF) content. An integrated transcriptome-assisted label-free quantitative proteomic approach by High Definition Mass Spectrometry (HDMSE) was employed to identify and quantify the proteins. A functional enrichment analysis using the differentially abundant proteins list was performed to understand the biological processes involved in IMF deposition. Moreover, to explore and clarify the biological mechanisms that influence IMF content, the mRNA data for the same trait from Cesar and collaborators [2] obtained by RNA-sequencing technology was compared with proteomic data. The mRNA data is deposited in the European Nucleotide Archive (ENA) repository (EMBL-EBI), under accession PRJEB13188.

Longissimus dorsi muscle label-free quantitative proteomic reveals biological mechanisms associated with intramuscular fat deposition.

The pathways involved in intramuscular fat (IMF) deposition in Longissimus dorsi muscle were investigated using an integrated transcriptome-assisted label-free quantitative proteomic approach by High Definition Mass Spectrometry. We quantified 1582 proteins, of which 164 were differentially abundant proteins (DAPs, p < 0.05) between animals with high (H) and low (L) genomic estimated breeding values (GEBV) for IMF content. Ingenuity pathway analysis (IPA) revealed that these DAPs were mainly involved in glycolysis metabolism, actin cytoskeleton signaling, cell-cell adherens junction and pathways for MAPK and insulin. A comparative study between transcriptomic (mRNA) and proteomic data showed 17 differentially expressed genes corresponding to DAPs, of which three genes/proteins did not agree on the direction of the fold change between groups. Moreover, we investigated microRNAs data to explain these differences in fold change direction, being able to unravel two of the three unexpected mRNA/protein relationships. Results demonstrated that changes in protein/mRNA levels of sarcomere organization, intracellular signal transduction and regulation of actin cytoskeleton, are involved in IMF deposition. These findings provide a deeper understanding of the highly complex regulatory mechanisms involved in IMF deposition in cattle and indicate target pathways for future studies. SIGNIFICANCE: Intramuscular fat is the amount of fat deposited inside muscle and plays an important role in human health and meat quality attributes, influencing energy metabolism of skeletal muscle, as well as, tenderness, flavor, and juiciness of beef. We performed for the first time the utilization of integrated transcriptome-assisted label-free quantitative proteomic approach using High Definition Mass Spectrometry for characterization of the changes in the proteomic profile of the Longissimus dorsi muscle associated with intramuscular fat deposition in cattle. Furthermore, we compared the muscle proteome with the muscle transcriptome (mRNA and microRNAs), obtained by RNA-sequencing, to better understand the relationship between expression of mRNAs and proteins and to unravel essential biological mechanisms involved in bovine skeletal muscle IMF deposition.

Differential expression of albumins and globulins of wheat flours of different technological qualities revealed by nanoUPLC-UDMSE.

Soluble proteins were extracted from common wheat flour obtained from nine cultivars of different qualities and were analyzed by nanoUPLC and Ultra Definition Mass Spectrometry (UDMSE) label-free quantitative approach. Collectively, 5894 proteins were identified and quantified with 8 peptides/protein. A total of 414 proteins were found differentially expressed with 85% proteins not yet described in the literature, according to their biological function. Quality-related proteins, such as puroindolines and chaperones, notably involved in the gluten-protein folding process, were up-regulated in superior (SP) and medium (MD) flours qualities and down-regulated in the low (LW) ones. Domains related to the chaperones activity were also found up-regulated in SP and MD and down-regulated in LW. In this work, nanoUPLC-UDMSE analysis was effective to identify and quantify metabolic proteins, allowing a clear distinction of the wheat flours and providing new perspectives for evaluating the technological quality based on the low abundant proteins of wheat endosperm.

Differential Metabolism of a Two-Carbon Substrate by Members of the Paracoccidioides Genus.

The genus Paracoccidioides comprises known fungal pathogens of humans and can be isolated from different infection sites. Metabolic peculiarities in different members of the Paracoccidioides led us to perform proteomic studies in the presence of the two-carbon molecule acetate, which predominates in the nutrient-poor environment of the phagosome. To investigate the expression rates of proteins of different members of Paracoccidioides, including one isolate of P. lutzii (Pb01) and three isolates of P. brasiliensis (Pb03, Pb339, and PbEPM83), using sodium acetate as a carbon source, proteins were quantified using label-free and data-independent liquid chromatography-mass spectrometry. Protein profiles of the isolates were statistically analyzed, revealing proteins that were differentially expressed when the fungus was cultivated in a non-preferential carbon source rather than glucose. A total of 1,160, 1,211, 1,280, and 1,462 proteins were reproducibly identified and relatively quantified in P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively. Notably, 526, 435, 744, and 747 proteins were differentially expressed among P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively, with a fold-change equal to or higher than 1.5. This analysis revealed that reorganization of metabolism occurred through the induction of proteins related to gluconeogenesis, glyoxylic/glyoxylate cycle, response to stress, and degradation of amino acids in the four isolates. The following differences were observed among the isolates: higher increases in the expression levels of proteins belonging to the TCA and respiratory chain in PbEPM83 and Pb01; increase in ethanol production in Pb01; utilization of cell wall components for gluconeogenesis in Pb03 and PbEPM83; and increased ß-oxidation and methylcitrate cycle proteins in Pb01and PbEPM83. Proteomic profiles indicated that the four isolates reorganized their metabolism in different manners to use acetate as a carbon source.

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host.

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity.

Acanthoscurria gomesiana is a Brazilian spider from the Theraphosidae family inhabiting regions of Southeastern Brazil. Potent antimicrobial peptides as gomesin and acanthoscurrin have been discovered from the spider hemolymph in previous works. Spider venoms are also recognized as sources of biologically active peptides, however the venom peptidome of A. gomesiana remained unexplored to date. In this work, a MS-based workflow was applied to the investigation of the spider venom peptidome. Data-independent and data-dependent LC-MS/MS acquisitions of intact peptides and of peptides submitted to multiple enzyme digestions, followed by automated chromatographic alignment, de novo analysis, database and homology searches with manual validations showed that the venom is composed by <165 features, with masses ranging from 0.4-15.8kDa. From digestions, 135 peptides were identified from 17 proteins, including three new mature peptides: U1-TRTX-Agm1a, U1-TRTX-Agm2a and U1-TRTX-Agm3a, containing 3, 4 and 3 disulfide bonds, respectively. The toxins U1-TRTX-Agm1a differed by only one amino acid from U1-TRTX-Ap1a from A. paulensis and U1-TRTX-Agm2a was derived from the genicutoxin-D1 precursor from A. geniculata. These toxins have potential applications as antimicrobial agents, as the peptide fraction of A. gomesiana showed activity against Escherichia coli, Enterobacter cloacae and Candida albicans strains. MS data are available via ProteomeXchange Consortium with identifier PXD003884. BIOLOGICAL SIGNIFICANCE: Biological fluids of the Acanthoscurria gomesiana spider are sources of active molecules, as is the case of antimicrobial peptides and acylpolyamines found in the hemolymphs. The venom is also a potential source of toxins with pharmacological and biotechnological applications. However, to our knowledge no A. gomesiana venom toxin structure has been determined to date. Using a combination of high resolution mass spectrometry, transcriptomics and bioinformatics, we employed a workflow to fully sequence, determine the number of disulfide bonds of mature peptides and we found new potential antimicrobial peptides. This workflow is suitable for complete peptide toxin sequencing when handling limited amount of venom samples and can accelerate the discovery of peptides with potential biotechnological applications.

Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity

Acanthoscurria gomesiana is a Brazilian spider from the Theraphosidae family inhabiting regions of Southeastern Brazil. Potent antimicrobial peptides as gomesin and acanthoscurrin have been discovered from the spider hemolymph in previous works. Spider venoms are also recognized as sources of biologically active peptides, however the venom peptidome of A. gomesiana remained unexplored to date. In this work, a MS-based workflow was applied to the investigation of the spider venom peptidome. Data-independent and data-dependent LC-MS/MS acquisitions of intact peptides and of peptides submitted to multiple enzyme digestions, followed by automated chromatographic alignment, de novo analysis, database and homology searches with manual validations showed that the venom is composed by <165 features, with masses ranging from 0.4-15.8 kDa. From digestions, 135 peptides were identified from 17 proteins, including three new mature peptides: U1-TRTX-Agm1a, U1-TRTX-Agm2a and Ul-TRTX-Agm3a, containing 3, 4 and 3 disulfide bonds, respectively. The toxins U1-TRTX-Agm1a differed by only one amino acid from Ul-TRTX-Apl a from A. paulensis and U1-TRTX-Agm2a was derived from the genicutoxin-D1 precursor from A. geniculata. These toxins have potential applications as antimicrobial agents, as the peptide fraction of A. gomesiana showed activity against Escherichia coli, Enterobacter cloacae and Candida albicans strains. MS data are available via ProteomeXchange Consortium with identifier PXD003884. Biological significance: Biological fluids of the Acanthoscurria gomesiana spider are sources of active molecules, as is the case of antimicrobial peptides and acylpolyamines found in the hemolymphs. The venom is also a potential source of toxins with pharmacological and biotechnological applications. However, to our knowledge no A. gomesiana venom toxin structure has been determined to date. Using a combination of high resolution mass spectrometry, transcriptomics and bioinformatics, we employed a workflow to fully sequence, determine the number of disulfide bonds of mature peptides and we found new potential antimicrobial peptides. This workflow is suitable for complete peptide toxin sequencing when handling limited amount of venom samples and can accelerate the discovery of peptides with potential biotechnological applications.

Proteomics in quality control: Whey protein-based supplements.

UNLABELLED: The growing consumption of nutritional supplements might represent a problem, given the concern about the quality of these supplements. One of the most used supplements is whey protein (WP); because of its popularity, it has been a target of adulteration with substitute products, such as cheaper proteins with lower biological value. To investigate this type of adulteration, this study used shotgun proteomics analyses by MS(E) (multiplexed, low- and high-collision energy, data-independent acquisition) of WP-based supplements. Seventeen WP-based supplement samples were evaluated. Chicken, maize, rice, potato, soybean, and wheat proteins were considered as probable sources of bovine whey adulteration. Collectively, 523 proteins were identified across all 16 samples and replicates, with 94% of peptides inside a normal distribution within 10ppm of maximum error. In 10 of the 16 samples analyzed, only proteins from bovine whey could be detected, while in the other samples several other protein sources were detected in high concentrations, especially soybean, wheat, and rice. These results point out a probable adulteration and/or sample contamination during manufacturing that could only be detected using this proteomic approach. SIGNIFICANCE: The present work shows how shotgun proteomics can be used to provide reliable answers in quality control matters, especially focusing on Whey Protein nutritional supplements which are a very popular subject in food and nutrition. In order to achieve an appropriate methodology, careful evaluation was performed applying extremely rigorous quality criteria, established for the proteomic analysis. These criteria and the methodological approach used in this work might serve as a guide for other authors seeking to use proteomics in quality control.

Baseline resolution of isomers by traveling wave ion mobility mass spectrometry: investigating the effects of polarizable drift gases and ionic charge distribution.

We have studied the behavior of isomers and analogues by traveling wave ion mobility mass spectrometry (TWIM-MS) using drift-gases with varying masses and polarizabilities. Despite the reduced length of the cell (18 cm), a pair of constitutional isomers, N-butylaniline and para-butylaniline, with theoretical collision cross-section values in helium (ΩHe ) differing by as little as 1.2 Å(2) (1.5%) but possessing contrasting charge distribution, showed baseline peak-to-peak resolution (Rp-p ) for their protonated molecules, using carbon dioxide (CO2), nitrous oxide (N2O) and ethene (C2H4 ) as the TWIM drift-gas. Near baseline Rp-p was also obtained in CO2 for a group of protonated haloanilines (para-chloroaniline, para-bromoaniline and para-iodoaniline) which display contrasting masses and theoretical ΩHe , which differ by as much as 15.7 Å(2) (19.5%) but similar charge distributions. The deprotonated isomeric pair of trans-oleic acid and cis-oleic acid possessing nearly identical theoretical ΩHe and ΩN2 as well as similar charge distributions, remained unresolved. Interestingly, an inversion of drift-times were observed for the 1,3-dialkylimidazolium ions when comparing He, N2 and N2O. Using density functional theory as a means of examining the ions electronic structure, and He and N2-based trajectory method algorithm, we discuss the effect of the long-range charge induced dipole attractive and short-range Van der Waals forces involved in the TWIM separation in drift-gases of differing polarizabilities. We therefore propose that examining the electronic structure of the ions under investigation may potentially indicate whether the use of more polarizable drift-gases could improve separation and the overall success of TWIM-MS analysis.