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Hair is a noninvasive valuable biospecimen for the long-term assessment of endogenous metabolic disturbance. Whether the hair is suitable for identifying biomarkers of the Alzheimer’s disease (AD) process remains unknown. We aim to investigate the metabolism changes in hair after β-amyloid (Aβ1-42) exposure in rats using ultra-high-performance liquid chromatography-high-resolution mass spectrometry–based untargeted and targeted methods. Thirty-five days after Aβ1-42 induction, rats displayed significant cognitive deficits, and forty metabolites were changed, of which twenty belonged to three perturbed pathways: (1) phenylalanine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis—l-phenylalanine, phenylpyruvate, ortho-hydroxyphenylacetic acid, and phenyllactic acid are up-regulated; (2) arachidonic acid (ARA) metabolism—leukotriene B4 (LTB4), arachidonyl carnitine, and 5(S)-HPETE are upregulation, but ARA, 14,15-DiHETrE, 5(S)-HETE, and PGB2 are opposite; and (3) unsaturated fatty acid biosynthesis— eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), FA 18:3 + 1O, and FA 18:3 + 2O are downregulated. Linoleic acid metabolism belonging to the biosynthesis of unsaturated fatty acid includes the upregulation of 8-hydroxy-9,10-epoxystearic acid, 13-oxoODE, and FA 18:2 + 4O, and downregulation of 9(S)-HPODE and dihomo-γ-linolenic acid. In addition, cortisone and dehydroepiandrosterone belonging to steroid hormone biosynthesis are upregulated. These three perturbed metabolic pathways also correlate with cognitive impairment after Aβ1-42 stimulation. Furthermore, ARA, DHA, EPA, l-phenylalanine, and cortisone have been previously implicated in the cerebrospinal fluid of AD patients and show a similar changing trend in Aβ1-42 rats’ hair. These data suggest hair can be a useful biospecimen that well reflects the expression of non-polar molecules under Aβ1-42 stimulation, and the five metabolites have the potential to serve as novel AD biomarkers.

Compared with the rapid advances in genomics leading to broad understanding of human disease, the linkage between chemical exposome and diseases is still under investigation. High-resolution mass spectrometry (HRMS) is expected to accelerate the process via relatively accurate and precise biomonitoring of human exposome. This review covers recent advancements in biomonitoring of exposed environmental chemicals (chemical exposome) using HRMS described in the 124 articles that resulted from a systematic literature search on Medline and Web of Science databases. The analytical strategic aspects, including the selection of specimens, sample preparation, instrumentation, untargeted versus targeted analysis, and workflows for MS-based biomonitoring to explore the environmental chemical space of human exposome, are deliberated. Applications of HRMS in human exposome investigation are presented by biomonitoring (1) exposed chemical compounds and their biotransformation products; (2) DNA/protein adducts; and (3) endogenous compound perturbations. Challenges and future perspectives are also discussed.

The current approaches remain insufficient for measuring chicken egg spoilage or present analytical limitations. This study aimed to complement the existing analyses and identify novel markers using liquid chromatography-high resolution mass spectrometry-based foodomics strategies. In the discovery set, comparative untargeted metabolomics was utilized to identify marker candidates in microbially inoculated chicken eggs. Markers were annotated by spectral matching with authentic standards, experimental libraries, or in silico fragmentation. In the validation set, targeted metabolomics was employed to verify the markers in stored chicken eggs from five farms. Statistical differences at a p-value < 0.001 revealed increases in lactic and 3-hydroxybutyric acids and decreases in phosphocholine, LPE(O-18:1), LPC(16:0), and LPC(18:0) in stored eggs. Receiver operating characteristic curve analysis of the six combined markers yielded an AUC of 0.956 and a sensitivity and specificity of ∼90%. Four phospholipids were highlighted as a novel class of spoilage markers. Our findings may contribute to further industrial implementation, benefiting the quality assurance and food safety of poultry egg production.

Aged black garlic (BG) is a functional food in global markets; however, very few studies have ventured into comprehensive profiling of BG metabolomes during the aging process. Herein, we exploited UHPLC-Orbitrap HRMS for a comparative metabolomics analysis. During the heat treatment, organosulfur compounds such as allicin, diallyl disulfide, ajoene, S-allyl-l-cysteine (SAC), and γ-glutamyl-SAC were downregulated. Plenty of glycerophospholipids together with shikimate, aromatic amino acids, and vitamin B6 vitamers were significantly augmented; tryptophan was however consumed to generate downstream products manifested in nicotinate metabolism and aminobenzoate degradation. These secondary metabolites serve as signaling mediators or protectants against extreme thermal exposure. Besides, Heyns compounds and Amadori-rearrangement byproducts with potential mutagenic effects were concentrated. Together, our findings expand the known metabolome space of BG processing and better elucidate the reactivities of the key metabolites. We provide in-depth insights into the biochemical changes of BG that enable further functional or toxicological investigations of this popular food.

Fish among other food can easily become spoilage. However, the existing literature endeavouring into a holistic investigation of fish metabolome during storage is scarce. There is a need for a powerful tool for more in-depth determinations. The present study is leveraging high-resolution mass spectrometry (HRMS)-based untargeted foodomics in the exploration of potential indicators for fish freshness. Three batches of fish fillets were stored in the refrigerator (+4 °C) for 0, 24, 48, and 72 h. Features were detected via UPLC-Q-Orbitrap HRMS and hereby undergone selection, identification, and verification. Eight sensitive indicators with significant time-dependent responses were yielded. The loss of freshness in fish is manifested in the upregulation of uracil, hypoxanthine, and inosine (involved in nucleotide changes) and α-linolenic acid, docosahexaenoic acid, arachidonic acid, and linoleic acid (involved in lipid hydrolysis) as well as in the downregulation of decanoylcarnitine (involved in fatty acid metabolism). Our work provides a promising approach to assess the quality of fish during storage and gain deeper insights into the metabolic reaction.

Background: Alzheimer’s disease (AD) is a common and progressive neurodegenerative disorder related to cognitive impairments that generally include deficits in short-term memory. Hair may be a potential biospecimen for AD biomarker discovery since it reflects the integral metabolic profiles of body burden over several months. Here, we discovered hair biomarkers in hair that differentiate AD patients from cognitively healthy controls using a high-resolution mass spectrometry (HRMS)-based untargeted metabolomics approach.Methods: An HRMS-based untargeted metabolomics approach was used to discover the potential AD biomarker candidates in hair samples from 24 AD patients (including n = 14, 7, and 3, with CDR scores of 0.5, 1, and 2, respectively) and 24 age- and sex-matched cognitively healthy controls. The hair samples were collected 0.1-cm away from the scalp and further cut into 3-cm segments. Hair metabolites were extracted by ultrasonication with methanol/phosphate-buffered saline 50/50 (v/v) for 4 hours. We assessed metabolic biomarker to distinguish between AD and controls with Student’s t test, hierarchical cluster analysis (HCA), and receiver operating characteristic (ROC) curve analysis.Results: Statistically significant changes in 61 peak features in hair between the patients with AD and cognitively healthy controls were discovered, of which 25 were identified to have chemical structures. The levels of nine metabolites, including acetyl-L-carnitine, propionylcarnitine, butyrylcarnitine, O-valeroyl-L-carnitine, PC (16:0/0:0), LPC 18:1, piperine, hydroxyprolyl-leucine, and 6-O-methylnorlaudanosoline, were significantly lower in patients with questionable AD (CDR score of 0.5) than in cognitively healthy controls (p < 0.01). ROC curves were constructed to evaluate the performance of the nine metabolites combined into a metabolic marker panel for distinguishing patients with questionable AD from cognitively healthy controls, in which the sensitivity and specificity were 86% and 71%, respectively, and the AUC value was 0.85 (95% CI: 0.72~0.97), indicating high potential for the initiation of AD dementia.Conclusions: Nine metabolites may be used as biomarkers for the early detection of AD dementia or therapeutic development after validation in a larger cohort. The present study suggested that the hair metabolome can be used to reveal metabolic perturbations for biomarker discovery and may lead to the development of a sensitive diagnostic tool for AD.

Conjugation reactions are of critical significance in human metabolism. Identification of these conjugated metabolites is still challenging. Here, we propose a strategy, post-deconvolution MS/MS spectra extraction with data-independent acquisition (PDMS2E-DIA), to comprehensively profile the glucuronide-conjugated metabolome. PDMS2E-DIA enables the identification of conjugated and unconjugated metabolite pairs through neutral loss filtering combined with a significant change in abundance after the deconjugation reaction. Purified DIA MS/MS spectra were constructed by extracting MS/MS fragments shared between spectra derived from conjugated and unconjugated metabolites. The feasibility of this approach was first demonstrated by the identification of two glucuronide-conjugated metabolite standards spiked in urine samples. For human urine samples, 479 features were structurally annotated as potential glucuronide-conjugated metabolites, resulting in the identification of 211 metabolites. Fragment peaks derived from interferents were found to be removed by PDMS2E-DIA, which increased about 6 times the number of identified urine metabolites compared with those calculated from raw DIA deconvoluted MS/MS spectra. This approach was found to have great potential for identifying glucuronide-conjugated metabolites, as well as other kinds of chemical conjugations.

The proteins of extracellular vesicles (EVs) that originate from tumors reflect the producer cells’ proteomes and can be detected in biological fluids. Thus, EVs provide proteomic signatures that are of great interest for screening and predictive cancer diagnostics. By applying targeted mass spectrometry with stable isotope-labeled peptide standards, we assessed the levels of 28 EV-associated proteins, including the conventional exosome markers CD9, CD63, CD81, CD82, and HSPA8, in vesicles derived from the lung cancer cell lines NCI-H23 and A549. Furthermore, we evaluated the detectability of these proteins and their abundance in plasma samples from 34 lung cancer patients and 23 healthy volunteers. The abundance of TLN1, TUBA4A, HSPA8, ITGB3, TSG101, and PACSIN2 in the plasma of lung cancer patients was measured using targeted mass spectrometry and compared to that in plasma from healthy volunteers. The most diagnostically potent markers were TLN1 (AUC, 0.95), TUBA4A (AUC, 0.91), and HSPA8 (AUC, 0.88). The obtained EV proteomic signature allowed us to distinguish between the lung adenocarcinoma and squamous cell carcinoma histological types. The proteomic cargo of the extracellular vesicles represents a promising source of potential biomarkers.

Phthalate esters are a group of synthetic industrial chemicals that are widely used in plastics. Urinary phthalate metabolites are short-term exposure markers frequently used to represent exposure levels in environmental epidemiology studies. Human hair is an alternative matrix for recording long-term exposure, but there are still analytical challenges that need to be addressed. In this study, an analytical method was established for simultaneously measuring nine major phthalate metabolites in human hair and successfully applied to measure phthalate metabolites in 30 hair samples collected from 30 individual human volunteers without known occupational exposure to phthalates. Two portions of 25 mg of hair samples were extracted by acidified methanol and water in 240 min of ultrasonication and then analyzed using a liquid chromatography-tandem mass spectrometry system. The limit of quantification ranged from 0.72 to 10.7 ng/g hair for nine phthalate metabolites. All nine phthalate metabolites were detected in more than 70% of the 30 individual human hair samples. The measured levels of hair phthalate metabolites were (in descending order): MEHP MMP ≫ MEP MBP (MnBP + MiBP) MiNP MEHHP ≈ MEOHP ≈ MECPP. The primary metabolite, MEHP (692 ± 582 ng/g), is the major DEHP metabolite in hair. This result is consistent with the findings in blood but not in urine, in which the secondary metabolites are the major DEHP metabolites. This method is easy to foresee with a clinical application and applies to human biomonitoring studies to assess long-term environmental phthalate exposure.

Di-(2-propylheptyl) phthalate (DPHP) is an alternative plasticizer that can replace other phthalates currently being scrutinized, and its use and production volumes are increasing. This study aimed to develop a high-resolution mass spectrometry (HRMS)-based metabolomics strategy to comprehensively screen urinary biomarkers of DPHP exposure and filter out potentially useful DPHP exposure markers for human exposure assessments. This strategy included three stages: screening of biomarkers, verification of dose-response relationships in laboratory animals, and application in human subjects. The multivariate data analysis method known as orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to screen and find meaningful signals in an MS dataset generated from urine samples collected from DPHP-administered rats. Thirty-six MS signals were verified as exposure marker candidates by assessing dose-response relationships in an animal feeding study. A biotransformation product of DPHP, mono-(2-propyl-7-dihydroxy-heptyl) phthalate, was suggested as a DPHP exposure marker for general human exposure assessments after the human application study and chemical structure identification. Three previously oxidized DPHP biotransformation products might be suitable for human exposure assessments in high-level exposure groups but not in the general population due to their low sensitivity. Keywords: Phthalate, Di-(2-propylheptyl)phthalate, Biomarker, Metabolomics, Exposure assessment

Diisononyl phthalate (DiNP) is commonly used as a plasticizer in polyvinyl chloride (PVC) products. However, DiNP can interfere with the human endocrine system and is listed as a chemical with reproductive toxicity. It has a short half-life and can be metabolized into water-soluble substances that are excreted in the urine, and thus, DiNP exposure markers can be identified in urine, a noninvasive and easily accessible specimen. In a previous study, we established an isotope tracing strategy involving an in-house computational signal mining algorithm with isotope tracing (SMAIT) to filter probable metabolite signals from complex LC-MS data. In this study, we used SMAIT to filter the probable metabolite signals, which were verified as potential DiNP exposure markers by analyzing human urine specimens from rubber workers who had occupational exposure to DiNP. We filtered 16 probable metabolite signals out of approximately 1400 signals by SMAIT. Fourteen of the 16 probable metabolite signals were confirmed as DiNP structure-related metabolites according to the fragment ion at m/z 121.029, which is a benzoate ion (C6H5CO2), identified in D0-MMOP mass spectra by ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) in the full-scan mode. The 14 structure-related metabolite signals were further verified as potential DiNP exposure markers by analyzing rubber worker urine specimens. Three DiNP structure-related metabolite signals at m/z 305.139 (MOINP), 307.155 (MHINP), and 375.142 (derivatives of MHINP) were identified as potential exposure markers of DiNP. We need more research to validate whether these metabolites could be used as specific biomarkers for human exposure to DiNP. The 3 structure-related metabolite signals could provide the basis for future exposure and risk assessment for DiNP.

Di-(2-propylheptyl) phthalate (DPHP) is a plasticizer and has been suggested to be a subchronic toxicant in rats. DPHP has been approved to be used in food containers and handling by the U.S. Food and Drug Administration. The use of DPHP is still increasing, and the risk of human exposure to DPHP via food may be high. Exposure markers measured in human samples are commonly used to monitor human exposure levels. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and a rat model were used to discover tentative DPHP exposure markers. DPHP and mono-(2-propylheptyl) phthalate (MPHP) were used as the precursors for calculating metabolite candidates using biotransformation mass changes of known enzymatic reactions. A rat model was designed to validate these metabolite candidates as tentative exposure markers. A total of 28 signals show dose–response relationships and these signals contain a few isomers. The chemical structures of 15 tentative exposure marker signals were speculated based on the product ion mass spectra from MS/MS analysis. These 15 signals included 7 chemical structures and some of them may be isomers. The different arrangement of the atoms in space of these isomers should be validated by standard compounds in the future studies. Among the 7 speculated chemical structures, 2 structures were novel tentative DPHP metabolites, and 5 structures have been previously reported in the literature. The results indicate that using UPLC-MS and a rat model can be used to identify tentative toxicant exposure markers. Keywords: Biotransformation mass changes, Di-(2-propylheptyl) phthalate, Ultra-performance liquid chromatography-mass spectrometry

—Using selected reaction monitoring (SRM) a targeted analysis of proteins that were previously reported to be associated with lung cancer (Novikova, S.E. et al., Biomeditsinskaya khimiya, 2017, vol. 63, pp. 181−210 [1]) was carried out. Measurements were performed using: 23 samples of plasma of patients with lung adenocarcinoma (LAC), 11 samples of plasma of patients with squamous cell lung carcinoma (SqCC), 25 samples of healthy donors. Analysis of plasma protein concentrations has shown that among 52 proteins registered by SRM statistically significant changes were found only for 8 proteins; five of them increased in the group of lung cancer patients (P00738, P26639, P21926, P08603, P51149), while plasma concentrations of three proteins (P51884, O15162, Q8N2K0) were lower than in control. Comparison of plasma protein concentrations in lung cancer patients did not reveal statistically significant changes between LAC and SqCC. These potential biomarkers can be used for \development of methods for early diagnostics of lung cancer.

Over recent years, metabolomics has been featured as the state-of-the-art technology that successfully opens the paths to understanding biological mechanisms and facilitating biomarker discovery. However, the inherent dynamic and sensitive nature of the metabolome have been challenging the accuracy of capturing the timepoints of interest while using biofluids such as urine and blood. Hair has thus emerged as a valuable analytical specimen for the long-term and retrospective determinations. Unfortunately, notwithstanding the apparent interest on global hair metabolomics, very few studies have engaged in the optimisation of the extraction strategy. In this study, we systemically investigated the extraction procedures for hair metabolome using a single factor experimental design. Three pH values (acidic, neutral, and basic) in aqueous solution, six extraction solvents (methanol, acetonitrile, acetone, phosphate-buffered saline, deionised water, and dichloromethane), different compositions of selected solvent mixtures and their sequential extraction, and a series of extraction times (15, 45, 60, 120, 240, and 480 min) were evaluated. The ideal condition for hair extraction is ultrasonic-assisted extraction with methanol:phosphate-buffered saline 50:50 (v/v) under +55 °C for 240 min. This strategy may secure the true composition of the metabolome, maximise the signal abundance, and guarantee a high coverage of wide-range metabolites in a straightforward approach. The optimised extraction strategy was then coupled with structure annotation tools for hair metabolome profiling. After a single RPLC-HRMS run, hair metabolite identification was achieved as the annotations of 171 probable structures and 853 tentative structures as well as the assignments of 414 unequivocal molecular formulae. In conclusion, we established an efficient extraction strategy for untargeted hair metabolomics, which the method is deliverable to any analytical laboratories and the sample can be directly profiled by means of a conventional RPLC-HRMS gradient.

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is a plasticizer used in polyvinyl chloride (PVC) products, such as toys and food packaging. Because the use of DINCH is on the rise, the risk of human exposure to this chemical may likewise increase. Discovering markers for assessing human chemical exposure is difficult because the metabolism of chemicals within humans is complex. In this study, two mass spectrometry (MS)-based metabolite profiling data processing methods, the mass defect filter (MDF) method and the signal mining algorithm with isotope tracing (SMAIT) method, were used for DINCH metabolite discovery, and 110 and 18 potential DINCH metabolite signal candidates were discovered, respectively, from in vitro DINCH incubation samples. Of these, the 21 signals were validated as tentative exposure marker signals in a rat model. Interestingly, the two methods generated rather different sets of DINCH exposure markers. Five of the 21 tentative exposure marker signals were verified as the probable DINCH structure-related metabolite signals based on their MS/MS product ion profiles. These five signals were detected in at least one human urine sample. Of the five probable DINCH structure-related metabolite signals, two novel signals might be suitable exposure markers that should be further investigated for their application in human DINCH exposure assessments. These observations indicate that the MDF and SMAIT methods may be used to discover a relatively different set of potential DINCH exposure markers.

Di-(2-propylheptyl) phthalate (DPHP) is a plasticizer used in polyvinyl chloride and vinyl chloride copolymer that has been suggested to be a toxicant in rats and may affect human health. Because the use of DPHP is increasing, the general German population is being exposed to DPHP. Toxicant metabolism is important for human toxicant exposure assessments. To date, the knowledge regarding DPHP metabolism has been limited, and only four metabolites have been identified in human urine. Ultra-performance liquid chromatography was coupled with Orbitrap high-resolution mass spectrometry (MS) and two data-screening approaches-the signal mining algorithm with isotope tracing (SMAIT) and the mass defect filter (MDF)-for DPHP metabolite candidate discovery. In total, 13 and 104 metabolite candidates were identified by the two approaches, respectively, in in vitro DPHP incubation samples. Of these candidates, 17 were validated as tentative exposure biomarkers using a rat model, 13 of which have not been reported in the literature. The two approaches generated rather different tentative DPHP exposure biomarkers, indicating that these approaches are complementary for discovering exposure biomarkers. Compared with the four previously reported DPHP metabolites, the three tentative novel biomarkers had higher peak intensity ratios, and two were confirmed as DPHP hydroxyl metabolites based on their MS/MS product ion profiles. These three tentative novel biomarkers should be further investigated for potential application in human exposure assessment.

The full characterization of nonbiological complex drugs (NBCDs) is not possible, but analytical approaches are of urgent need to evaluate the similarity between different lots and compare with their follow-up versions. Here, we propose a hypothesis testing-based approach to assess the similarity/difference between random amino acid copolymer drugs using liquid chromatography mass spectrometry (LC-MS) analysis. Two glatiramer acetate (GA) drugs, commercially available Copaxone and in-house synthesized SPT, and a negative control were digested by Lys-C and followed by HILIC-MS analysis. After retention time alignment and feature identification, 1627 features matched to m/z values in an elemental composition database were considered as derived from active drug ingredients. A hypothesis testing approach, the sum of squared deviations test, was developed to process high-dimensional data derived from LC-MS spectra. The feasibility of this approach was first demonstrated by testing 5 versus 5 lots of Copaxone and Copaxone versus SPT, which suggested a significant similarity by obtaining the estimated 95th percentile of the distribution of the estimator (ρ(95%)) at 0.0056 (p-value = 0.0026) and 0.0026 (p-value < 0.0001), respectively. In contrast, the ρ was 0.036 (p-value = 1.00), while comparing Copaxone and the negative control, implying a lack of similarity. We further synthesized nine stable isotope-labeled peptides to validate the proposed amino acid sequences in the database, demonstrating the correctness in sequence identification. The quantitation variations in our analytical procedures were determined to be 6.8-7.7%. This approach was found to have a great potential for evaluating the similarity between generic NBCDs and listed reference drugs, as well as to monitor the lot-to-lot variation.

Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The outer membrane of exosomes protects their content from degradation and contains markers of the parent cell. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. Due to fact that exosomes contain proteins of cells secreting them, these vesicles could be a valuable source for biomarkers discovery. Currently, a number of studies prove the participation of exosomes in carcinogenesis. However, there is a problem of isolating pure and characterized exosomes for further use in investigation of functions or identification of tumor protein biomarkers. In this work, we have performed experiments on exosomes isolation from human plasma by three methods: differential ultracentrifugation, ultracentrifugation in sucrose cushion, sedimentation of the exosomal fraction from serum by using a commercial kit. The protein composition of the obtained samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The obtained exosomal samples were searched for the presence of exosomal markers (CD9, CD82, HSPA8, CD63). In the samples of exosomes isolated by ultracentrifugation with the sucrose cushion, the content of the above markers was determined as 32.85, 15.59, 6.07 fmol/mg of total protein, correspondently. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes.Ékzosomy – membrannye mikrovezikuly, vydeliaemye kletkami v biologicheskie zhidkosti. Membrana ékzosom zashchishchaet ikh soderzhimoe ot degradatsii i soderzhit markery produtsiruiushcheĭ ikh kletki. Ékzosomy produtsiruiutsia prakticheski vsemi kletkami organizma, kletki opukholeĭ sekretiruiut ikh osobenno sil'no. Odnako, pri issledovanii funktsiĭ étikh vnekletochnykh vezikul i identifikatsii opukholevykh belkovykh biomarkerov v ikh sostave na nachal'nom étape voznikaet ser'eznaia problema vydeleniia chistykh, okharakterizovannykh preparatov ékzosom. V dannoĭ rabote proveden kolichestvennyĭ proteomnyĭ analiz ékzosom syvorotki krovi cheloveka, poluchennykh pri pomoshchi differentsial'nogo ul'tratsentrifugirovaniia, ul'tratsentrifugirovaniia v gradiente plotnosti sakharozy ili osazhdeniia ékzosomal'noĭ fraktsii syvorotki s ispol'zovaniem kommercheskogo nabora Exosome Isolation Kit (“Invitrogen from ThermoFisher Scientific Baltics”, UAB, Litva). Mass-spektrometricheskimi metodami monitoringa vybrannykh reaktsiĭ (SRM) i panoramnogo mass-spektrometricheskogo analiza provedeno opredelenie belkovogo sostava vydelennykh ékzosom. Poluchennye preparaty byli okharakterizovany po soderzhaniiu osnovnykh markerov (CD9, CD82, HSPA8, CD63). V obraztsakh ékzosom, vydelennykh iz syvorotki zdorovykh dobrovol'tsev metodom ul'tratsentrifugirovaniia v gradiente plotnosti sakharozy, bylo opredeleno soderzhanie vysheukazannykh markerov, kotoroe sostavilo 32,85, 15,59, 6,07 fmol' na 1mkg belka preparata ékzosom, sootvetstvenno. Ostal'nye metody, vkliuchaia kommercheskiĭ nabor, ne dali polozhitel'nykh rezul'tatov. Rezul'taty étogo issledovaniia pokazyvaiut, chto metod tsentrifugirovaniia v gradiente plotnosti sakharozy iavliaetsia naibolee optimal'nym dlia vydeleniia ékzosom.

Analyzing and presenting data from multiple groups are much more informative than that from two groups. However, common tools such as S plot and volcano plot are only available for identifying the significant features between two groups and are restricted to multiple-group comparisons. This study proposed novel visualization plots which not only overcame the restrictions of the above methods but also utilized the p values of multiple tests as the x-axis. The novel visualization plots included a parametric method and a nonparametric method. The parametric method was a combination of an analysis of variance and Welch’s analysis of variance; the nonparametric method used the Kruskal-Wallis test. During the selection of significant features, machine learning algorithms were used to determine the cutting points of the x-axis. As a proof of concept, the real data from the experiments of 4-MeO-α-PVP metabolites and fish spoilage metabolomics were illustrated via our visualization method. The results showed that the novel visualization plots were much efficiently presented to identify significant metabolites in multiple-group comparisons. Especially, the positive predicted values of the nonparametric method and the cutting points determined by logistic regression were higher than those of other machine learning algorithms in determining the cutting points for multiple groups.

The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

Previous studies have suggested that kidney stone formers are associated with a higher risk of cardiovascular events. To our knowledge, there have been no previous examinations of the relationship between carotid intima-media thickness (IMT) and urinary stone risk factors. This study was aimed toward an investigation of the association between dyslipidemia, IMT, and 24-hour urinalysis in patients with calcium oxalate (CaOx) or calcium phosphate (CaP) stones. We prospectively enrolled 114 patients with kidney stones and 33 controls between January 2016 and August 2016. All patients were divided into four groups, according to the stone compositions&mdash, CaOx &ge, 50% group, CaP group, struvite group, and uric acid stones group. Carotid IMT and the carotid score (CS) were evaluated using extracranial carotid artery doppler ultrasonography. The results of a multivariate analysis indicated that a higher serum total cholesterol (TC) and low-density lipoprotein (LDL) were all associated with lower urinary citrate and higher CS in both the CaOx &ge, 50% and CaP groups. Higher serum TC and LDL were also associated with increased serum 8-OHdG levels in both groups. The levels of carotid IMT and CS in the CaOx &ge, 50% and CaP groups were all significantly higher than in the controls. These findings suggest a strong link between dyslipidemia, carotid atherosclerosis, and calcium kidney stone disease.

Purpose Plasma markers that enable diagnosis in the early stage of lung cancer is not discovered. A liquid chromatography multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay for identifying potential early marker proteins for lung adenocarcinoma is developed. Experimental design LC-MRM-MS assay is used for measuring the level of 35 candidate peptides in plasma from 102 lung adenocarcinoma patients (including n = 50, 16, 24, and 12 in stage I, II, III, and IV, respectively.) and 84 healthy controls. Stable isotope labeled standard peptides are synthesized to accurately measure the amount of these proteins. Results Seven proteins are able to distinguish stage I patients from controls. These proteins are combined in to a protein marker panel which improve the sensitivity to discriminate stage I patients from controls with cross-validated area under the curve = 0.76. Besides, it is found that low expression of eukaryotic initiation factor 4A-I and high expression of lumican show significantly poor prognosis in overall survival (p = 0.012 and 0.0074, respectively), which may be used as prognostic biomarkers for lung cancer. Conclusions and clinical relevance Proteins highlighted here may be used for early detection of lung adenocarcinoma or therapeutics development after validation in a larger cohort.

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.

In the present study, we applied selected reaction monitoring (SRM) to a group of proteins that were previously reported to be associated with lung cancer (Novikova S.E. et al. (2017) Biomeditsinskaya khimiya, 63, 181-210. [1]). Measurements were performed on 59 plasma samples. These samples included: 23 samples of plasma of patients diagnosed with lung adenocarcinoma (LAC), 11 samples of plasma of patients diagnosed with squamous cell lung carcinoma (SqCC), 25 samples of donors with no previous history of oncological diseases, and one pooled sample from each of the above group. As a result of the SRM measurements 52 proteins were detected at least in one individual plasma sample. Statistical analysis showed that there were two groups confidently differentiated by the concentration value of 8 proteins wherein 5 proteins displayed increased level (P00738, P26639, P21926, P08603, P51149) in LAC group and 3 proteins (P51884, O15162, Q8N2K0) indicated diminishing the concentration level towards the control level. Data on protein concentrations obtained for LAC and SqCC did not distinguish the samples by statistical clustering analysis. These potential biomarkers can be used for further development of methods for early diagnostics of lung cancer.Metodom monitoringa vybrannykh reaktsiĭ (MVR) proveden targetnyĭ kolichestvennyĭ analiz belkov, dlia kotorykh ranee soobshchalos' ob ikh sviazi s razvitiem raka legkogo (Novikova S.E. i dr. (2017) Biomeditsinskaia khimiia, 63, 181-210. [1]). Izmereniia provodili v probakh plazmy krovi 25 obraztsov zdorovykh donorov i 34 patsientov s diagnozom nemelkokletochnyĭ rak legkikh (23 obraztsa s diagnozom adenokartsinomy legkogo (LAC), 11 – ploskokletochnaia kartsinoma legkikh (SqCC)). Analiz poluchennykh znacheniĭ kontsentratsii belkov pokazal, chto iz 52 zaregistrirovannykh metodom MVR v plazme krovi belkov statisticheski znachimo razlichaiutsia kontsentratsii 8 belkov: dlia 5-i nabliudaetsia povyshenie urovnia ékspressii (P00738, P26639, P21926, P08603, P51149) u bol'nykh, a dlia 3 belkov snizhenie kontsentratsii (P51884, O15162, Q8N2K0) po sravneniiu s kontrolem. Sravnenie znacheniĭ kontsentratsiĭ belkov obraztsov plazmy LAC i SqCC ne vyiavilo dostovernykh razlichiĭ v kontsentratsii belkov pri étikh zabolevaniiakh. Obnaruzhennye potentsial'nye biomarkery mogut sluzhit' dlia razrabotki podkhodov ranneĭ diagnostiki.

Urine and hair are used for assessing human exposure to toxicants. Urine tests can show acute toxicant exposure. Hair analysis can be used to determine chronic toxicant exposure after months to years; however, compared to urine, hair analysis in exposure assessments is much less frequently investigated. Urine and hair are different matrices, and their mechanisms of toxicant metabolite incorporation are different. The toxicant metabolites present in urine and hair may also be different. To clarify this issue, a procedure was developed to identify toxicant metabolites in rat samples using a mass spectrometry-based metabolomic data processing method. Di-(2-propylheptyl) phthalate (DPHP), an industrial plasticizer, was used as the model toxicant. The developed procedure identified not only known DPHP metabolites (mono-(propyl-6-oxo-heptyl) phthalate, mono-(propyl-6-hydroxyheptyl) phthalate, and mono-(propyl-6-carboxyhexyl) phthalate) but also novel metabolites that were structurally related to DPHP in the rat samples, indicating that the developed procedure successfully identified toxicant metabolites in in vivo samples. Among the 62 tentative metabolites identified from the 7th-day urine and the 28th-day hair samples, 33 were detected in only the urine samples, 19 were detected in only the hair samples, and 10 were identified in both the urine and hair samples. A total of 15 out of the 62 metabolites were confirmed as DPHP structure-related metabolites based on MS/MS analysis. Among the 15 DPHP structure-related metabolites, only 2 metabolites were present in both the urine and hair samples. These results suggested that the metabolites identified in urine could not be applied to exposure assessments based on hair analysis.

BackgroundDifferential expression and secretion of alpha-actinin 4 (ACTN4) in the lung cancer cell lines CL1-0 and CL1-5 have been reported in previous proteomic studies. The aim of this study is to investigate the functional properties of the ACTN4 protein in non–small-cell lung cancer (NSCLC) cells and evaluate its clinical importance.MethodsWe used RNA interference to knock down and overexpress ACTN4 protein to evaluate the effects of this intervention on cancer cell invasion and migration, as well as on microscopic cellular morphology. Furthermore, we examined by immunohistochemistry the expression of ACTN4 protein in tissue samples at different stages of lung cancerand compared the protein levels of ACTN4 in blood plasma samples from patients with histologically confirmed lung cancer and healthy controls.ResultsCL1-5 cell motility was significantly suppressed by the knockdown of ACTN4 protein. The morphology of CL1-5 cells changed from a predominantly mesenchymal-like shape into a globular shape in response to ACTN4 protein knockdown. A quantitative immunohistochemical assessment of lung cancer tissues revealed that ACTN4 protein level was considerably higher in cancerous tissues than in the adjacent normal ones, and the area under the receiver operating characteristic curve was 0.736 (p < 0.001). According to an enzyme-linked immunosorbent assay, the plasma levels of ACTN4 protein were significantly different between cancer patients and healthy controls, and the areas under the receiver operating characteristic curves were 0.828 and 0.909, respectively, for two independent cohorts (p < 0.001).ConclusionsWe demonstrate that the knockdown of ACTN4 protein inhibited cell invasion and migration. These results suggest that ACTN4 is associated with lung cancer cell motility. Thus, the level of ACTN4 in cancerous tissue and plasma is related to the presence of lung cancer.

Tissue inhibitor metalloproteinase-1 (TIMP-1) is clinically associated with a poor prognosis for various cancers, but the roles of TIMP-1 in lung cancer metastasis are controversial. Our previous secretomic study revealed that TIMP-1 is highly abundant in high invasiveness cells of lung adenocarcinoma. In the current study, TIMP-1 abundances in primary lung adenocarcinoma tissues, as revealed by immunohistochemistry, are significantly higher in patients with lymph invasion and distant metastasis than in those without. Receiver operating characteristic curve analyses suggest 73.7 and 86.2 % accuracy to separate patients with lymph node and distant metastasis and those without, respectively. Moreover, we demonstrate that the expression level of TIMP-1 positively associates with cell mobility, invasiveness, and metastatic colonization. Most notably, the novel mechanism in which TIMP-1 facilitates metastatic colonization through the mediation of pericellular polyFN1 assembly was revealed. In summary, this study presents novel functions of TIMP-1 in promoting cancer metastasis and suggests TIMP-1 is a potential tissue biomarker for lymph invasion and distant metastasis of lung adenocarcinoma.

Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation.

Streptococcus pyogenes is responsible for various diseases. During infection, bacteria must adapt to adverse environments, such as the acidic environment. Acidic stimuli may stimulate S. pyogenes to invade into deeper tissue. However, how this acidic stimulus causes S. pyogenes to manipulate its secretome for facilitating invasion remains unclear. The dynamic label-free LC–MS/MS profiling identified 97 proteins, which are influenced by environmental acidification. Among these, 33 (34%) of the identified proteins were predicted to be extracellular proteins. Interestingly, classical secretory proteins comprise approximately 90% of protein abundance of the secretome in acidic condition at the stationary phase. One acid-induced secreted protein, HtpA, was selected to investigate its role in invasive infection. The mouse infected by the htpA deficient mutant showed lower virulence and smaller lesion area than the wild-type strain. The mutant strain was more efficiently cleared at infected skin than the wild-type strain. Besides, the relative phagocytosis resistance is lower in the mutant strain than in the wild-type strain. These data indicate that a novel acid-induced virulence factor, HtpA, which improves anti-phagocytosis ability for causing necrotizing fasciitis. Our investigation provides vital information for documenting the broad influences and mechanisms underlying the invasive behavior of S. pyogenes in an acidified environment. Biological significance The acidified infected environment may facilitate S. pyogenes invasion from the mucosa to the deeper subepithelial tissue. The acid stimuli have been considered to affect the complex regulatory network of S. pyogenes for causing severe infections. Many of secreted virulence factors influenced by acidified environment may also play a crucial role in pathogenesis of invasive disease. To investigate temporal secretome changes under acidic environment, a comparative secretomics approach using label-free LC–MS/MS was undertaken to analyze the secretome in acidic and neutral conditions. The dynamic label-free LC–MS/MS profiling and secretome prediction were used in this study for mining acid-influenced secreted proteins. We identified 33 acid-influenced secreted proteins in this study. Among these proteins, a novel acid-induced virulence factor, HtpA, was demonstrated to improve anti-phagocytosis ability for causing necrotizing fasciitis. In addition, our study demonstrates the first evidence that acidic stimuli and growth-phase cues are crucial for classical protein secretion in S. pyogenes.

Objectives The objective of the study was to investigate the presence and distribution of endogenous steroids in human stratum corneum (SC) with respect to sex, age, anatomical site, and depth into SC, using a noninvasive sampling technique and a sensitive analytic method for quantitation. Materials and methods Corneocytes in the SC samples removed by sequential tape stripping from the forearm, forehead, and back sites were processed and analyzed using a validated liquid chromatography–tandem mass spectrometry method for the quantitation of hydrocortisone, cortisone, and testosterone. Results In the 32 volunteers surveyed, testosterone was only detected at the forearm site in a single volunteer. Both hydrocortisone and cortisone were detected in 5–7 individuals out of 16 from both the age 20–35 years and age 50–65 years groups. Maximal amounts of hydrocortisone and cortisone found in SC of forehead, forearm, and back were 0.37 ng/cm 2 , 0.96 ng/cm 2 , and 0.49 ng/cm 2 ; and 0.20 ng/cm 2 , 0.12 ng/cm 2 , and 0.06 ng/cm 2 , respectively, and were either higher than or comparable to those reported in human hair in terms of concentration by SC weight. In the population with either hydrocortisone or cortisone detected, no significant differences relating to sex, age groups, and anatomical sites were observed for the amount of hydrocortisone and cortisone in the SC. However, significantly higher amount of cortisone was found in the surface layers of SC than deeper layers in the age 50–65 years group. Conclusion The results demonstrate that, with the achievable sensitivity of current analytical technology, physiological concentrations of endogenous steroids, such as hydrocortisone and cortisone, can be found in the SC of some individuals.