EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides.

De novo sequencing of any novel peptide/protein is a difficult task. Full sequence coverage, isomeric amino acid residues, inter- and intramolecular S-S bonds, and numerous other post-translational modifications make the investigators employ various chemical modifications, providing a variety of specific fragmentation MSn patterns. The chemical processes are time-consuming, and their yields never reach 100%, while the subsequent purification often leads to the loss of minor components of the initial peptide mixture. Here, we present the advantages of the EThcD method that enables establishing the full sequence of natural intact peptides of ranid frogs in de novo top-down mode without any chemical modifications. The method provides complete sequence coverage, including the cyclic disulfide section, and reliable identification of isomeric leucine/isoleucine residues. The proposed approach demonstrated its efficiency in the analysis of peptidomes of ranid frogs from several populations of Rana arvalis, Rana temporaria, and Pelophylax esculentus complexes.

GC × GC-HRMS with complementary ionization methods in the suspect screening analysis of fragrance allergens: overwhelming or justified?

Modern gas chromatography-mass spectrometry (GC-MS) allows for the analysis of complex samples, such as fragrances. However, identifying all the constituents in natural fragrance mixtures, especially allergens that need to be listed on product labels, is a significant challenge. This is primarily due to the high complexity of the sample and the fact that electron ionization, the most commonly used ionization method in GC-MS, produces numerous nonspecific fragment ions, often resulting in the absence or very low abundance of the molecular ion. These factors affect confidence in assigning the analyte. In this study, we demonstrate that the combination of GC × GC separation, with high mass resolution and accurate mass measurements, as well as chemical ionization in addition to traditional electron ionization, becomes an efficient tool for reliable qualitative analysis of a mixture containing 100 fragrance allergens, even when many of them are closely related species or isomers. The proposed approach expands the applicability of the comprehensive GC × GC-HRMS method, which includes complementary ionization techniques, from studies on anthropogenic priority pollutants and emerging contaminants to the analysis of natural products. Although targeted qualitative and quantitative analysis of allergens in the modern laboratories is well organized, GC × GC-HRMS, being a useful complement to routine quality control of volatile allergens in fragrances, definitely gives an additional contribution to the analytical cases when conventional 1D-GC-MS faces some problems or uncertainties.

Mucolytic Drugs Ambroxol and Bromhexine: Transformation under Aqueous Chlorination Conditions.

Bromhexine and ambroxol are among the mucolytic drugs most widely used to treat acute and chronic respiratory diseases. Entering the municipal wastewater and undergoing transformations during disinfection with active chlorine, these compounds can produce nitrogen- and bromine-containing disinfection by-products (DBPs) that are dangerous for aquatic ecosystems. In the present study, primary and deep degradation products of ambroxol and bromhexine obtained in model aquatic chlorination experiments were studied via the combination of high-performance liquid and gas chromatography with high-resolution mass spectrometry. It was shown that at the initial stages, the reactions of cyclization, hydroxylation, chlorination, electrophilic ipso-substitution of bromine atoms with chlorine, and oxidative N-dealkylation occur. Along with known metabolites, a number of novel primary DBPs were tentatively identified based on their elemental compositions and tandem mass spectra. Deep degradation of bromhexine and ambroxol gives twenty-four identified volatile and semi-volatile compounds of six classes, among which trihalomethanes account for more than 50%. The specific class of bromhexine- and ambroxol-related DBPs are bromine-containing haloanilines. Seven of them, including methoxy derivatives, were first discovered in the present study. One more novel class of DBPs associated with bromhexine and ambroxol is represented by halogenated indazoles formed through dealkylation of the primary transformation products containing pyrazoline or tetrahydropyrimidine cycle in their structure.

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.

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.

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.

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) [...].

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.

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.

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.

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.

Gas-phase study of the stability of α-substituted cyclic amino nitriles under electron ionization and electrospray ionization and fragmentation peculiarities of cyclic ketimines.

RATIONALE: α-Substituted cyclic amino nitriles show different reactivity and stability in solution depending on their ring size. Mass-spectrometric modeling of the reactions, which normally take place in solution, may shed light on the nature of the studied compounds and explain some observations. METHODS: The stability of the nitriles in an acidic solution was studied by adding glacial acetic acid. Gas-phase modelling was conducted by gas chromatography/mass spectrometry (GC/MS) with electron ionization mass spectrometry and direct inlet electrospray ionization. QqQ and Orbitrap analyzers were used to carry out tandem mass spectrometry (MS/MS) experiments. RESULTS: The obtained data show that the elimination of HCN from α-substituted cyclic amino nitriles occurs in the same way for an acidic solution, thermolysis and electrospray ionization. According to GC, the most stable were N-formylated nitriles compared with N-benzylated or N-unsubstituted ones. Electrospray ionization demonstrated its advantages for the routine identification of cyclic amino nitriles due to milder conditions than in electron ionization. Fragmentations of cyclic ketimines, formed from N-unsubstituted amino nitriles, are discussed in particular. CONCLUSIONS: Similarities in the behavior of the α-substituted cyclic amino nitriles under electron ionization, electrospray ionization, thermolysis and reaction in solution under acidic catalysis were discovered and confirmed by MS/MS experiments. Fragmentation schemes of the studied nitriles and corresponding imines are proposed.

Water/Alkali-Catalyzed Reactions of Azides with 2-Cyanothioacetamides. Eco-Friendly Synthesis of Monocyclic and Bicyclic 1,2,3-Thiadiazole-4-carbimidamides and 5-Amino-1,2,3-triazole-4-carbothioamides.

The reactions of thioamides with azides in water were studied. It was reliably shown that the reaction of 2-cyanothioacetamides 1 with various types of azides 2 in water in the presence of alkali presents an efficient, general, one-step, atom-economic, and eco-friendly method for the synthesis of 1,2,3-thiadiazol-4-carbimidamides 5 and 1,2,3-triazole-4-carbothioamides 4. This method can be extended to the one-pot reaction of sulfonyl chlorides and 6-chloropyrimidines 2'o with sodium azide, leading to final products in higher yields, that is, avoiding the isolation of unsafe sulfonyl azides. The method was furthermore applied to the reaction of N,N'-bis-(2-cyanothiocarbonyl)pyrazine 1h with sulfonyl azides to afford bicyclic 1,2,3-thiadiazoles 8 and 1,2,3-triazoles 9 connected via a 1,1'-piperazinyl linker. 2-Cyanothioacetamides 1 were also shown to react with aromatic azides in water in the presence of alkali to afford 1-aryl-5-amino-1,2,3-triazole-4-carbothioamides 11. In contrast to aromatic azides and similarly to sulfonyl azides, 6-azidopyrimidine-2,4-diones 2o-q react with cyanothioacetamides to form N-pyrimidin-6-yl-5-dialkylamino-1,2,3-thiadiazole-4-N-l-carbimidamides 12. A mechanism was proposed to rationalize the role of water in changing the reactivity of azides toward 2-cyanothioacetamides.

Switchable Synthesis of 4,5-Functionalized 1,2,3-Thiadiazoles and 1,2,3-Triazoles from 2-Cyanothioacetamides under Diazo Group Transfer Conditions.

High yield solvent-base-controlled, transition metal-free synthesis of 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-triazoles from 2-cyanothioacetamides and sulfonyl azides is described. Under diazo transfer conditions in the presence of a base in an aprotic solvent 2-cyanothioacetamides operating as C-C-S building blocks produce 5-amino-4-cyano-1,2,3-thiadiazoles exclusively. The use of alkoxide/alcohol system completely switches the reaction course due to the change of one of the reaction centers in the 2-cyanothioacetamide (C-C-N building block) resulting in the formation of 5-sulfonamido-1,2,3-triazole-4-carbothioamide sodium salts as the only products. The latter serve as good precursors for 5-amino-1,2,3-thiadiazole-4-carboximidamides, the products of Cornforth-type rearrangement occurring in neutral protic medium or under acid conditions. According to DFT calculations (B3LYP/6-311+G(d,p)) the rearrangement proceeds via intermediate formation of a diazo compound, and can be catalyzed by acids via the protonation of oxygen atom of the sulfonamide group.

Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes.

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

Exploration of doubtful cases of leucine and isoleucine discrimination in mass spectrometric peptide sequencing by electron-transfer and higher-energy collision dissociation-based method.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z2-ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation.

RATIONALE: Mass spectrometry has shown itself to be the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most efficient methods of triggering fragmentation and combine all the possible complementary techniques. METHODS: Collision-induced dissociation (CID), high-energy collision dissociation (HCD), and electron-transfer dissociation (ETD) tandem mass spectra recorded with a LTQ Orbitrap Velos instrument were used for the elucidation of the sequence of the natural non-tryptic peptides from the skin secretion of Rana latastei. Manual interpretation of the spectra was applied. RESULTS: The combined approach using CID, HCD, and ETD tandem mass spectra of the multiprotonated peptides in various charge states, as well as of their proteolytic fragments, allowed the sequences of seven novel peptides from the skin secretion of Rana latastei to be established. CONCLUSIONS: Manual mass spectrometry sequencing of natural non-tryptic peptides from the skin secretion of Rana latastei provided the opportunity to work successfully with these species and demonstrated once again its advantage over automatic approaches.

Proteolytic degradation and deactivation of amphibian skin peptides obtained by electrical stimulation of their dorsal glands.

Amphibians are among the oldest creatures on our planet. Their only defensive weapon efficient against microorganisms and predators involves their skin secretion. The wide range of biological activities of the peptides in the skin secretion of amphibians makes these compounds rather interesting for generation of prospective pharmaceuticals. The first step in studying these molecules requires their structures to be established. Mass spectrometry is the most powerful tool for this purpose. The sampling and sample preparation stages preceding mass spectrometry experiments appear to be rather crucial. The results obtained here demonstrate that these preparation procedures might lead to partial or complete loss of the bioactive peptides in the secretion. Five minutes in water was enough to completely destroy all of the bioactive peptides in the skin secretion of the marsh frog (Rana ridibunda); even immediate addition of methanol to the water solution of the peptides did not prevent partial destruction. Concerted effort should be directed towards development of the most efficient procedure to keep the secreted peptides intact. Graphical Abstract ᅟ.