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.

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.

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.

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.

EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond.

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides.

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.

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.

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.

Discrimination of leucine and isoleucine in peptides sequencing with Orbitrap Fusion mass spectrometer.

An efficient approach to easy and reliable differentiation between isomeric leucine and isoleucine in peptide sequencing utilizes multistage electron transfer dissociation and higher energy collision activated dissociation in the Orbitrap Fusion mass spectrometer. The MS(3) method involves production and isolation of primary odd-electron z(•) ions, followed by radical site initiation of their fragmentation with formation of w-ions, characteristic of the isomeric amino acid residues. Six natural nontryptic peptides isolated from the secretion of frog Rana ridibunda were studied. Their lengths were in the range between 15 and 37 amino acids and the number of targeted isomeric (Leu/Ile) residues varied between 1 and 7. The experiments were successful in all 22 cases of Leu/Ile residues, leaving no doubts in identification. The method is extremely selective as the targeted w-ions appear to be the most intense in the spectra. The proposed approach may be incorporated into shotgun proteomics algorithms and allows for the development of an exclusively mass spectrometric method for automated complete de novo sequencing of various peptides and proteins.

N-terminal tagging strategy for de novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS.

The major portion of skin secretory peptidome of the European Tree frog Hyla arborea consists of short peptides from tryptophyllin family. It is known that b-ions of these peptides undergo head-to-tail cyclization, forming a ring that can open, resulting in several linear forms. As a result, the spectrum contains multiple ion series, thus complicating de novo sequencing. This was observed in the Q-TOF spectrum of one of the tryptophyllins isolated from Hyla arborea; the sequence FLPFFP-NH(2) was established by Edman degradation and counter-synthesis. Though no rearrangements were observed in FTICR-MS and MALDI-TOF/TOF spectra, both of them were not suitable for mass-spectrometry sequencing due to the low sequence coverage. To obtain full amino acid sequence by mass spectrometry, three chemical modifications to N-terminal amino moiety were applied. They include acetylation and sulfobenzoylation of N-amino group and its transformation to 2,4,6-trimethylpyridinium by interaction with 2,4,6-trimethylpyrillium tetrafluoroborate. All three reagents block scrambling and provide spectra better than the intact peptide. Unfortunately, all of them also readily react with lysine side chain. Hence, all investigated procedures can be used to improve sequencing of short peptides, while acetylation is the recommended one. It shows excellent results, and it is plain and simple to perform. This is the procedure of choice for MS-sequencing of short peptides by manual or automatic algorithms.

Two dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures.

A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.

De novo sequencing of peptides secreted by the skin glands of the Caucasian Green Frog Rana ridibunda.

Amphibian skin glands are known to secrete various types of bioactive peptides. The array of these peptides is specific for every frog species. The present research deals with the identification of peptides isolated from the skin secretion of the Marsh frog R. ridibunda inhabiting the Kolkhida Canyon of the Caucasian region. The research is based on comprehensive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of intact and chemically modified peptides. In particular, an oxidation procedure was applied directly to the crude skin secretion to open S--S loops whereas N-terminal acetylation was additionally carried out for one individual peptide. Sequences were determined by manual interpretation of electron capture dissociation (ECD) and collisionally induced dissociation (CID) tandem mass spectra. A total of 29 peptides were identified in the skin secretion of the Caucasian Marsh frog. The peptide profile is represented with disulfide-containing peptides belonging to the brevinin, esculentin and ranatuerin families, neuropeptides of the bradykinin and bombesin families. Two identified peptides belonging to the ranatuerins are the first peptides of this family discovered in the skin secretions of European frogs. Ten of the identified peptides coincide with those reported earlier for the European Edible frog. Another ten are identical to those found in R. ridubunda from the Moscow region. This fact verifies the described method as being an efficient analytical tool to compare intra- and interspecific variabilities.