Mass Spectrometric De Novo Sequencing of Natural Peptides.

Natural peptides secreted under stress conditions by many organisms are bioactive molecules with a broad spectrum of activities. These molecules could become potential models for novel pharmaceuticals, to which bacteria, according to modern scientific concepts, do not have and cannot develop resistance. Taking this into consideration, it is necessary to clarify the amino acid sequences of such peptides. Here we describe our approach to de novo sequencing of amphibians' skin secretion peptides.

Cocamidopropyl betaine - a potential source of nitrogen-containing disinfection by-products in pool water.

Water treatment for most public pools involves disinfection with active chlorine leading to the formation of disinfection by-products (DBPs). Among them, nitrogen-containing compounds (N-DBPs) having increased toxicity and adverse effects on human health are of the greatest concern. Being the major component of various body washers for swimmers, cocamidopropyl betaine (CAPB) represents a potential and still underestimated anthropogenic precursor of N-DBPs in pool water. The purpose of this study was to investigate CAPB transformation pathways and mechanisms under the aqueous chlorination conditions. High-performance liquid and two-dimensional gas chromatography hyphenated with high-resolution mass spectrometry were used for the search and tentative identification of the primary and final CAPB transformation products. A wide range of DBPs containing up to five chlorine atoms including these in combination with hydroxyl and additional carbonyl groups has been revealed in model chlorination experiments for the first time. The proposed mechanism of their formation involves nucleophilic substitution of the secondary amide hydrogen atom at the first stage with subsequent free radical and electrophilic addition reactions resulting in non-selective introduction of halogen atoms and hydroxyl groups in the alkyl chain. The deep transformation products include short-chain chlorinated hydrocarbons and their oxidation products as well as dimethylcarbamoyl chloride possessing high toxicity and carcinogenic properties. Targeted analysis of real swimming pool water samples confirmed the results of model experiments enabling semi-quantitative determination of CAPB (0.8 µg L-1) and 18 primary DBPs, including 10 chlorine-containing compounds with the total concentration of 0.1 µg L-1. Among them, monochloro (50%) and hydroxydichloro (25%) derivatives predominate. The toxicity and health of the main DBPs has been estimated using QSAR/QSTR approach. Thus, the possibility of formation of new classes of potentially toxic chlorine-containing DBPs associated with the widespread use of detergents and cosmetics was shown.

Kinetic constants and transformation products of ornidazole during ozonation.

Ornidazole (ONZ), a nitroimidazole antibiotic detected in water bodies, may negatively impact the aquatic ecosystem. Its reaction kinetics during ozonation which is a feasible and applicable technology to control the contamination of emerging contaminants, however, has not been reported in literature. In this study, we measured the apparent second-order kinetic constant of ONZ with ozone molecules via the excessive ozone method and the competing method which led to an average value of 103.8 ± 2.7 M-1 s-1 at pH 7. The apparent second-order kinetic constant of ONZ with HO• was calculated to be 4.65 × 109 M-1 s-1 with the concept of Rct measured via para-chlorobenzoic acid as a probe. The transformation products (TPs) of ONZ during ozonation at pH 3 and pH 11 were separately analyzed with HPLC-MS/MS and some unique products were found at pH 11, reflecting the influence of HO•. The toxicity of individual TPs was predicted with the tool of T.E.S.T. It was found that 62% of 21 identified TPs could be more toxic than ONZ in terms of at least one acute toxicity endpoint, including chlorinated amines and N-oxides. The analysis with a respirometer further revealed that the toxicity of mixing TPs generated at HO• rich conditions was slightly lower than O3 dominated conditions. In general, this study provides the basic kinetic data for designing ozonation processes to eliminate ONZ and the important reference for understanding the toxicity evolution of ONZ during ozonation.

Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

EThcD Benefits for the Sequencing Inside Intramolecular Disulfide Cycles of Amphibian Intact Peptides.

Disulfide bonds formed by a pair of cysteine residues in the peptides' backbone represent a certain problem for their sequencing by means of mass spectrometry. As a rule, in proteomics, disulfide bonds should be cleaved before the analysis followed by some sort of chemical derivatization. That step is time-consuming and may lead to losses of minor peptides of the analyzed mixtures due to incomplete reaction, adsorption on the walls of the vials, etc. Certain problems in the de novo top-down sequencing of amphibian skin peptides are caused by the C-terminal disulfide loop, called the Rana box. Its reduction with or without subsequent derivatization was considered to be an unavoidable step before mass spectrometry. In the present study, EThcD demonstrated its efficiency in sequencing intact disulfide-containing peptides without any preliminary derivatization. Applied to the secretion of three frog species, EThcD provided the full sequence inside the intramolecular disulfide cycle for all S-S-containing peptides found in the samples, with the only exception being diarginine species. Proteolytic fragments, which are shorter than the original peptides, were helpful in some cases. HCD should be mentioned as a complementary tool to the EThcD tool, being useful as a confirmation method for some sequence details.

Differentiation between Isomeric 4,5-Functionalized 1,2,3-Thiadiazoles and 1,2,3-Triazoles by ESI-HRMS and IR Ion Spectroscopy.

A large variety of 1,2,3-thiadiazoles and 1,2,3-triazoles are used extensively in modern pure and applied organic chemistry as important structural blocks of numerous valuable products. Creation of new methods of synthesis of these isomeric compounds requires the development of reliable analytical tools to reveal the structural characteristics of these novel compounds, which are able to distinguish between isomers. Mass spectrometry (MS) is a clear choice for this task due to its selectivity, sensitivity, informational capacity, and reliability. Here, the application of electrospray ionization (ESI) with ion detection in positive and negative modes was demonstrated to be useful in structural studies. Additionally, interconversion of isomeric 4,5-functionalized 1,2,3-triazoles and 1,2,3-thiadiazoles was demonstrated. Application of accurate mass measurements and tandem mass spectrometry in MS2 and MS3 modes indicated the occurrence of gas-phase rearrangement of 1,2,3-triazoles into 1,2,3-thiadiazoles under (+)ESI-MS/MS conditions, independent of the nature of substituents, in line with the reaction in the condensed phase. Infrared multiple photon dissociation (IRMPD) spectroscopy enabled the establishment of structures of some of the most crucial common fragment ions, including [M+H-N2]+ and [M+H-N2-RSO2]+ species. The (-)ESI-MS/MS experiments were significantly more informative for the sulfonyl alkyl derivatives compared to the sulfonyl aryl ones. However, there was insufficient evidence to confirm the solution-phase transformation of 1,2,3-thiadiazoles into the corresponding 1,2,3-triazoles.

Global assessment of chemical quality of drinking water: The case of trihalomethanes.

BACKGROUND: Trihalomethanes (THM), a major class of disinfection by-products, are widespread and are associated with adverse health effects. We conducted a global evaluation of current THM regulations and concentrations in drinking water. METHODS: We included 120 countries (∼7000 million inhabitants in 2016), representing 94% of the world population. We searched for country regulations and THM routine monitoring data using a questionnaire addressed to referent contacts. Scientific and gray literature was reviewed where contacts were not identified or declined participation. We obtained or estimated annual average THM concentrations, weighted to the population served when possible. RESULTS: Drinking water regulations were ascertained for 116/120 (97%) countries, with 89/116 (77%) including THM regulations. Routine monitoring was implemented in 47/89 (53%) of countries with THM regulations. THM data with a varying population coverage was obtained for 69/120 (58%) countries consisting of ∼5600 million inhabitants (76% of world's population in 2016). Population coverage was ≥90% in 14 countries, mostly in the Global North, 50-89% in 19 countries, 11-49% among 21 countries, and ≤10% in 14 countries including India, China, Russian Federation and Nigeria (40% of world's population). DISCUSSION: An enormous gap exists in THM regulatory status, routine monitoring practice, reporting and data availability among countries, especially between high- vs. low- and middle-income countries (LMICs). More efforts are warranted to regulate and systematically assess chemical quality of drinking water, centralize, harmonize, and openly report data, particularly in LMICs.

Solving the mystery of the Chukotka stinky gray whales.

Gray whales (Eschrichtius robustus) constitute an important part of the diet of Chukotka Native population, reaching 30% of consumed food for the inland Chukchas. Over one hundred licenses for whale hunting are issued on an annual basis. After the USSR collapse natives had to hunt whales near the shore from the small boats. The problem of "stinky" whales arose immediately, as the meat of some harvested species possessed a strong medicinal/chemical odour. The hypotheses explaining the phenomenon ranged from biotoxins, to oil spills. To understand the problem, various tissues of normal and stinky Gray whales were collected in 2020-2021 and analyzed using headspace solid phase microextraction with Gas Chromatography - Mass Spectrometry. Here, we show that dozens of smelly organic compounds were identified among over 500 compounds detected in the samples. The most interesting analytes related to the off odour are bromophenols. The most probable suspect is 2,6-dibromophenol with strong iodoformic odour, perfectly matching that of the "stinky" whales. Quantitative results demonstrated its levels were up to 500-fold higher in the "stinky" whales' tissues. The source of 2,6-dibromophenol is likely polychaetes, producing 2,6-dibromophenol and colonising near shore waters where whales feed. Therefore, the mystery of the stinky whales may be considered resolved.

Mass Spectrometry Differentiation between Rana arvalis Populations Based on Their Skin Peptidome Composition.

Skin secretion of amphibians often represents the only weapon of these species against pathogens and predators. Peptides constitute the major portion of active molecules of that weapon and may be treated as potential pharmaceuticals for future generations. The first step of their efficient use involves establishing of their primary structure, i.e., sequencing. De novo sequencing by means of mass spectrometry was applied to Rana arvalis species, collected in the spring 2021 in Central Slovenia (vicinity of Ljubljana). HPLC-ESI-HRMS/MS with Orbitrap instruments was used to establish the skin peptidome of these species and compare it with the earlier identified skin peptidome of the Moscow population of Rana arvalis. Application of CID, HCD, ETD, and EThcD enabled detecting and sequencing 18 peptides; five of them were novel and may be treated as possible biomarkers of the Ljubljana population of Rana arvalis. Interestingly, representatives of two peptide families (temporins and brevinins 2) were not found in the Moscow population. MS3 modes, first of all EThcD, demonstrated their great potential in the de novo sequencing, including extraction of the sequence information from the intact peptides with disulfide cycle (rana box) in their structure and differentiation of isomeric Leu/Ile residues. Thus, all six isomeric residues were reliably distinguished in the novel melittin-related peptide AK-23-1. In addition, another post-translational modification dealing with carbonylation of the N-terminal Gly of novel temporin AVa was established using the MS3 mode. The obtained results demonstrate the efficiency of the use of MS3 tools in proteomics/peptidomics.

Aqueous Chlorination of D-Limonene.

Limonene (1-methyl-4-(1-methylethenyl)-cyclohexene) is one of the most widespread monocyclic terpenes, being both a natural and industrial compound. It is widely present in the environment, including in water supplies. Therefore, it may be subjected to aqueous chlorination at water treatment stations during drinking water preparation. Besides, being a component of numerous body care and cosmetic products, it may present at high levels in swimming pool waters and could also be subjected to aqueous chlorination. Laboratory experiments with aqueous chlorination of D-limonene demonstrated the prevalence of the conjugated electrophilic addition of HOCl molecule to the double bonds of the parent molecule as the primary reaction. The reaction obeys the Markovnikov rule, as the levels of the corresponding products were higher than those of the alternative ones. Fragmentation pattern in conditions of electron ionization enabled the assigning of the structures for four primary products. The major products of the chlorination are formed by the addition of two HOCl molecules to limonene. The reactions of electrophilic addition are usually accompanied by the reactions of elimination. Thus, the loss of water molecules from the products of various generations results in the reproduction of the double bond, which immediately reacts further. Thus, a cascade of addition-elimination reactions brings the most various isomeric polychlorinated species. At a ratio of limonene/active chlorine higher than 1:10, the final products of aqueous chlorination (haloforms) start forming, while brominated haloforms represent a notable portion of these products due to the presence of bromine impurities in the used NaOCl. It is worth mentioning that the bulk products of aqueous chlorination are less toxic in the bioluminescence test on V. fischeri than the parent limonene.

Urinary phthalate metabolite concentrations during four windows spanning puberty (prepuberty through sexual maturity) and association with semen quality among young Russian men.

AIM: To prospectively investigate the associations of urinary phthalate metabolite concentrations measured at four time points spanning pubertal development with semen parameters in Russian men. DESIGN: 516 boys were enrolled at ages 8-9 years (2003-2005) and followed annually. METHODS: Urine samples were collected annually and pooled into four exposure windows [prepuberty, early puberty, late puberty and sexual maturity] based on physician assessed Tanner genitalia stages and testicular volume. Fifteen phthalate metabolites were quantified using isotope dilution HPLC-MS/MS at Moscow State University. We calculated molar sums (∑) of di-2-ethylhexyl phthalate (DEHP), di-isononyl phthalate (DiNP), di-isodecyl phthalate (DiDP) and anti-androgenic phthalate (AAP) metabolites. At sexual maturity (ages 18-19 years), the men provided 1-2 semen samples for analysis. We estimated the associations of quintiles of urinary ∑phthalate metabolites as well as mono-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), and mono-benzyl phthalate (MBzP) at each pubertal window, with semen parameters by fitting generalized linear mixed models with random intercepts and adjusting for confounders. RESULTS: A total of 223 men who provided semen samples had phthalates measured at one or more pubertal windows. Higher urinary concentrations of ∑DiNP metabolites during late puberty were related to poorer semen quality (men with the highest quintile of urinary ∑DiNP had 30% lower sperm concentration, 32% lower count and 30% lower progressive motile count, compared to men in the lowest quintile). Also, young men with higher urinary concentrations of MiBP metabolites in early puberty tended to have poorer semen quality. No associations were observed for ∑DEHP metabolites, ∑DiDP metabolites, ∑AAP, MBzP or MnBP metabolites with semen quality parameters. CONCLUSIONS: ∑DiNP metabolites measured during late puberty and MiBP metabolites at early puberty were related to poorer semen quality, highlighting the importance of considering specific windows of exposure when investigating chemical exposures in relation to measures of reproductive health in men.

Associations of prepubertal urinary phthalate metabolite concentrations with pubertal onset among a longitudinal cohort of boys.

BACKGROUND: Although phthalate exposures have been associated with adverse effects on male reproductive health, few studies have explored longitudinal associations with male pubertal development. OBJECTIVES: We examined the association of prepubertal urinary concentrations of phthalate metabolites with age at pubertal onset in a prospective cohort of Russian boys. METHODS: At enrollment at ages 8-9 years, medical history, dietary, and demographic information was collected. At entry and annually, physical examinations and pubertal staging [Genitalia (G), Pubarche (P), and testicular volume (TV, in ml)] were conducted and spot urines were collected. Prepubertal urine samples (defined as either TV = 1, 2 and G = 1, 2 or TV = 3 and G = 1) were pooled for each boy and phthalate metabolite concentrations were quantified using isotope dilution LC-MS/MS at Moscow State University. We measured 15 metabolites including those from anti-androgenic parent phthalates (AAPs) such as di (2-ethylhexyl) (DEHP) and di-isononyl (DiNP) phthalates as well as monobenzyl (MBzP), mono-n-butyl (MnBP), and mono-isobutyl (MiBP) metabolites. We calculated the molar sums of DEHP (∑DEHP), DiNP (∑DiNP), and AAP (∑AAP) metabolites. Separate interval-censored models were used to assess associations of quartiles of prepubertal phthalate metabolites with each pubertal onset indicator, G2+, P2+ and TV > 3 mL, adjusted for covariates and urine specific gravity. RESULTS: 304 boys had 752 prepubertal urine samples (median 2, range: 1-6) for pooling. In adjusted models, higher urinary AAPs were consistently associated with later pubertal onset (P2) with mean shifts ranging from 8.4 to 14.2 months for the highest versus lowest quartiles. Significantly later onset for G2 and TV > 3 mL was observed for higher versus lower quartiles of MiBP, MBzP, ∑DEHP and ∑DiNP. CONCLUSIONS: On average, boys with higher concentrations of prepubertal urinary AAPs had later pubertal onset by six months to over a year. The impact of AAPs on timing of male puberty may be attributable to disruption of androgen-dependent biological pathways.

Planet Contamination with Chemical Compounds.

The number of known priority pollutants and emerging contaminants of environmental concern currently exceeds several thousand (US EPA Part 423, US EPA Part 401) [...].

The Sphingolipid Asset Is Altered in the Nigrostriatal System of Mice Models of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease incurable due to late diagnosis and treatment. Therefore, one of the priorities of neurology is to study the mechanisms of PD pathogenesis at the preclinical and early clinical stages. Given the important role of sphingolipids in the pathogenesis of neurodegenerative diseases, we aimed to analyze the gene expression of key sphingolipid metabolism enzymes (ASAH1, ASAH2, CERS1, CERS3, CERS5, GBA1, SMPD1, SMPD2, UGCG) and the content of 32 sphingolipids (subspecies of ceramides, sphingomyelins, monohexosylceramides and sphinganine, sphingosine, and sphingosine-1-phosphate) in the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of the preclinical and clinical stages of PD. It has been shown that in PD models, the expression of five of the nine studied genes (CERS1, CERS5, ASAH1, ASAH2, and GBA1) increases but only in the substantia nigra (SN) containing dopaminergic cell bodies. Changes in the expression of enzyme genes were accompanied by an increase in the content of 7 of the 32 studied sphingolipids. Such findings suggest these genes as attractive candidates for diagnostic purposes for preclinical and clinical stages of PD.

FT-MS in the de novo top-down sequencing of natural nontryptic peptides.

The present review covers available results on the application of FT-MS for the de novo sequencing of natural peptides of various animals: cones, bees, snakes, amphibians, scorpions, and so forth. As these peptides are usually bioactive, the animals efficiently use them as a weapon against microorganisms or higher animals including predators. These peptides represent definite interest as drugs of future generations since the mechanism of their activity is completely different in comparison with that of the modern antibiotics. Utilization of those peptides as antibiotics can eliminate the problem of the bacterial resistance development. Sequence elucidation of these bioactive peptides becomes even more challenging when the species genome is not available and little is known about the protein origin and other properties of those peptides in the study. De novo sequencing may be the only option to obtain sequence information. The benefits of FT-MS for the top-down peptide sequencing, the general approaches of the de novxxo sequencing, the difficult cases involving sequence coverage, isobaric and isomeric amino acids, cyclization of short peptides, the presence of posttranslational modifications will be discussed in the review.

Antiviral drug Umifenovir (Arbidol) in municipal wastewater during the COVID-19 pandemic: Estimated levels and transformation.

An indole derivative umifenovir (Arbidol) is one of the most widely used antiviral drugs for the prevention and treatment of COVID-19 and some other viral infections. The purpose of the present study was to shed light on the transformation processes of umifenovir in municipal wastewater, including disinfection with active chlorine, as well as to assess the levels of the antiviral drug and its metabolites entering and accumulating in natural reservoirs under conditions of the SARS-CoV-2 pandemic. The combination of high-performance liquid chromatography with electrospray ionization high-resolution mass-spectrometry and inductively coupled plasma mass spectrometry was used for tentative identification and quantification of umifenovir and its transformation products in model reaction mixtures and real samples of wastewater, river water, biological sludge and bottom sediments taken at the wastewater treatment plant in Arkhangelsk, a large cultural and industrial center at the Russian North. Laboratory experiments allowed identifying fifteen bromine-containing transformation products, forming at the initial stages of the chlorination and fourteen classic volatile and semi volatile disinfection by-products with bromoform as the dominant one. Chlorinated derivatives are only the minor disinfection by-products forming by substitution of alkylamine group in the aromatic ring. The schemes of umifenovir transformation in reactions with dissolved oxygen and sodium hypochlorite are proposed. Two established primary transformation products formed by oxidation of the thioether group to sulfoxide and elimination of thiophenol were detected in noticeable concentrations in the wastewater together with their precursor. The level of umifenovir reached 1.3 mg kg-1 in the sludge and municipal wastewater treat contained 1 µg L-1 of that drug, while its removal during biological wastewater treatment was about 40%. Pronounced accumulation of umifenovir and its transformation products in biological sludge and bottom sediments of natural reservoirs may be a source of the future secondary pollution of the environment.

Bioprospecting of Less-Polar Constituents from Endemic Brown Macroalga Fucus virsoides J. Agardh from the Adriatic Sea and Targeted Antioxidant Effects In Vitro and In Vivo (Zebrafish Model).

The endemic brown macroalga Fucus virsoides J. Agardh from the Adriatic Sea was in the focus of the present research. The volatiles of fresh (FrFv) and air-dried (DrFv) samples of F. virsoides obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were analyzed by gas chromatography equipped with flame ionization detector and mass spectrometry (GC-FID/MS). The major HS-FrFv compound was pentadecane (61.90-71.55%) followed by pentadec-1-ene (11.00-7.98%). In HS-DrFv, pentadec-1-ene was not present, and few lower aliphatic compounds appeared, as well as benzaldehyde and benzyl alcohol. In HD-FrFv, particularly abundant were alkenes (such as pentadec-1-ene (19.32%), or (E)-pentadec-7-ene (8.35%)). In HD-DrFv, more oxidation products were present (e.g., carbonyl compounds such as tridecanal (18.51%)). The fatty acids profile of freeze-dried sample (FdFv) after conversion to methyl esters was determined by GC-FID, and oleic acid was dominant (42.28%), followed by arachidonic acid (15.00%). High-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (HPLC-ESI-HRMS) was used for the screening of less polar fractions (F3 and F4) of F. virsoides. Mono- and diglycerides of stearic, palmitic, oleic, and arachidonic acids were found. Terpenoids and steroids comprised the compounds C20H30(32)O2 and C29H48O(2). Among carotenoids, fucoxanthin was identified. Chlorophyll derivatives were also found (C55H74(72)N4O(5-7)), dominated by pheophytin a. The antioxidant activity of the fractions was investigated by in vitro assays (oxygen radical absorbance capacity (ORAC), reduction of radical cation (ABTS•+), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, and ferric reducing antioxidant power (FRAP)) and by in vivo zebrafish model (along with fish embryotoxicity). In vitro experiments proved good radical scavenging abilities of F3 and F4 fractions, which were additionally supported by the protective effect against hydrogen peroxide-induced oxidative stress in zebrafish embryos.

Monitoring and Statistical Analysis of Formation of Organochlorine and Organobromine Compounds in Drinking Water of Different Water Intakes.

The main drawback of drinking water chlorination involves the formation of quite hazardous disinfection by-products (DBPs), represented mainly by halogenated species. Based on the authors' monitoring data since 2002, the prevalence of chlorine over bromine in the composition of volatile DBPs was shown for the drinking water in Ufa (Russia). However, the situation was completely reversed in the case of semi-volatile DBPs. The principal goal of the present study involved rationalization of the results of the long-term monitoring. Gas chromatography-mass spectrometry (GC-MS) was used for the qualitative and quantitative analysis of volatile DBPs. Identification of semi-volatile compounds was carried out with GC-MS, while gas chromatography with an atomic emission detector (GC-AED) was used for their quantification. A significant contribution of oxygen to the composition of semi-volatile compounds proves the decisive role of the dissolved organic matter oxidative destructive processes. Statistical analysis revealed notable linear correlations for trihalomethane and haloacetic acid formation vs. chlorine dose. On the contrary, halogenated semi-volatile products do not demonstrate any correlations with the water quality parameters or chlorine dose. Principal component analysis (PCA) placed them into separate groups. The results allow for proposing that formation of the organohalogenated species involved the fast penetration of bromine into the humic matter molecules and, further, their oxidative destruction by active chlorine.

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis.

RATIONALE: Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides. METHODS: Manual interpretation of complementary collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation. RESULTS: Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28. CONCLUSIONS: Mirror-symmetrical peptides are sequenced using N-terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b-ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.

Comprehensive two-dimensional gas chromatography-high resolution mass spectrometry with complementary ionization methods in the study of 5000-year-old mummy.

RATIONALE: Mummification is one of the defining customs of ancient Egypt. The nuances of the embalming procedure and the composition of the embalming mixtures have attracted the attention of scientists and laypeople for a long time. Modern analytical tools make mummy studies more efficient. METHODS: Comprehensive two-dimensional gas chromatography-high resolution mass spectrometry (GCxGC/HRMS) with complementary ionization methods (electron ionization, positive chemical ionization, and electron capture negative ionization [ECNI]) with a Pegasus GC-HRT+4D instrument was used to identify embalming components in the mummy from the Pushkin Museum of Fine Arts acquired in 1913 in London at the de Rustafjaell sale. The mummy dates back to the late Predynastic period (direct accelerator mass spectrometry-dating 3356-3098 bc), being one of the oldest in the world. RESULTS: The results showed the complexity of the embalming mixtures that were already in use 5000 years ago. Several hundred organic compounds were identified in the mummy samples. Various types of hydrocarbons (triterpanes, steranes, isoprenoid, and polycyclic aromatic hydrocarbons) prove the presence of petroleum products. Iodinated compounds detected using ECNI define oils of marine origin, whereas esters of palmitic acid indicate the use of beeswax. The nature of the discovered components of conifer tar proves that the preliminary processing of conifer resins involved heating. GCxGC/HRMS also allowed a number of modern contaminants (phthalates, organophosphates, and even DDT) to be identified. CONCLUSIONS: Application of a powerful GCxGC/HRMS technique with complementary ionization methods allowed significant widening of the range of organic compounds used for mummification that could be identified. The complexity of the embalming mixtures supports the hypothesis of the high social status of the child made on the basis of the preliminary study of the mummy.