Introduction

Experiencing stress, Anura amphibians release skin secretion with host-defense peptides (HDPs) as its major constituents [1, 2]. These molecules contribute to the innate and adaptive amphibian immunity. Complete array of these peptides forms skin peptidome which serves also as a taxonomy characteristic of anuran species [3], allowing for the reliable differentiation of closely related species and even populations [4]. Peptidomes of about 200 Anuran species (out of ~8000 inhabiting the Earth) are established so far [5, 6]. The corresponding peptides are divided into ~100 families [6, 7], named according to the amphibian species in which they were first discovered [3, 8].

HDPs are active against a wide range of bacteria, fungi, and protozoa. They also possess antiviral and antitumor activities [5, 9]. Recently, it was shown that HDPs participate in the frogs’ response on inflammation, influencing chemoattraction and thus accelerating wound healing [10, 11]. These facts make HDPs rather promising for the development of new generations of pharmaceuticals, required to fight microbes resistant to antibiotics [12]. Study of skin peptides is also important in understanding their role in the functioning of the entire organisms of frogs.

Brown frog Rana temporaria is the most widespread specie of ranid frogs in Europe. Not surprisingly, its peptidome was one of the first selected for studying. It is established with certain details but far from being completed, since the final result depends on the characteristics of the methods and instruments used [13,14,15,16,17,18]. Skin secretion of Rana temporaria contains peptides of the following families: disulfide-containing short (17–20 aa) brevinins 1 and long (33 aa) brevinins 2 [16, 17]; short (10–14 aa) amidated temporins [14, 18]; bradykinin and bradykinin-related peptides (BRPs) with numerous N- and C-extended copies [13, 15, 18], as well as melittin-related peptide (MRP-1) [16]. The most interesting in terms of pharmacology are, definitely, temporins [1, 19,20,21,22,23]. They are short, amidated, usually cationic peptides with amphipatic α-helix structure, active against a wide range of pathogens (gram-positive and gram-negative bacteria, protozoa, including Leishmania genus, viruses, fungi) [20, 24]. At the same time, they do not influence healthy mammalian cells. Some of them demonstrate hemotactyc activity, accelerating wound healing [25]. Some temporins may behave as anti-diabetic agents [22]. They are membranolytics interacting with the cell membrane by the mechanism avoiding resistance development by pathogens. They keep their properties in the blood plasma, while, being short, their synthesis is not so expensive. Moreover, some temporins are structurally similar to the peptides of mammalian brain and gastrointestinal tract [20].

Composition of skin peptidome of the common ranid species with wide habitat may vary to some extent [26, 27]. The changes involve certain peptides inside peptide families, but not the number of families in the peptidome. Thus, adaptation of the Slovenian population of the green frog Rana ridibunda resulted in the changes of composition of peptides inside families of brevinins 2 (6 novel peptides) and esculentins 1 and 2 (the number of these peptides decreased) [27].

The vast majority of Rana temporaria peptides were identified in the skin secretion, while the sequences of temporins B, G, and H were revealed by screening cDNA library of Rana temporaria [16]. Actually, transcriptome is often not identical to the skin peptidome due to uncorrelated fluctuations in transcriptome and peptide synthesis, as well as certain limitations of mass spectrometry: (i) losses of minor components of the secretion (sensitivity problems); (ii) instability of certain molecular species leading to the bad quality of the tandem mass spectra; (iii) joint elution of peptides and their adducts, interfering with the spectra interpretation [28, 29]. It is also worth mentioning (iv) posttranslational modifications and (v) fast degradation of bioactive peptides by endoproteases [30].

The main goal of our studies involves sequencing of natural non tryptic peptides of frogs’ skin secretion by means of mass spectrometry to reveal their peptidomes [30,31,32,33,34,35,36]. Skin secretions represent rather difficult for the direct analysis mixtures with a wide range of peptides. Some short peptides easily cyclize [37, 38], some peptides have symmetric N- and C-terminal sequences [39], and many of them have intramolecular S-S bonds [31, 40]. Moreover, some of them may have up to eight basic (Lys and Arg) amino acid residues in the chain. Trypsinolysis of the latter species results in formation of very short products, including even free amino acids. Taking into account the unknown genomes of the vast majority of frogs, application of bottom-up strategy in sequencing appears to be not very efficient. Therefore, to elucidate the sequence of novel HDPs, one should use top-down approach as the most efficient analytical tool. Fortunately, modern mass spectrometers, first of all Orbitraps and Fourier Transform Ion Cyclotron Resonance (FT ICR) MS instruments [33], allow for numerous variants of tandem mass spectra. Being accompanied with high mass accuracy, such mass spectra provide sufficient structural information for de novo sequencing of skin peptides. These instruments resolved the problem of the identification of isobaric residues (Lys/Gln; Phe/Metox; Tyr/Met2ox; etc.) and even the most difficult problem of mass spectrometry sequencing—distinguishing between the isomeric Leu and Ile residues [41,42,43].

The present work deals with comparative study of the composition of skin secretions of Rana temporaria species of Moscow (Russia) and Central Slovenian populations by means of complementary tandem mass spectrometry tools available with Orbitrap. The research aimed to sequence novel peptides and discover biomarkers of the frogs of Slovenian population.

Methods and materials

Reagents

The following reagents were used in the present study: acetonitrile, HPLC gradient grade (Sigma-Aldrich, St. Louis, MO, USA); formic acid, HPLC gradient grade (Fluca, Buchs, Switzerland). Water was prepared by Milli-Q water purification system (Millipore, Billerica, MA, USA).

Skin secretions

Two Rana temporaria frogs were caught in Central Slovenia (GKY: 459376, GKX: 111019, altitude: 317 m). Skin secretion was obtained by mild electrostimulation of their skin glands as described in [4]. The parameters of laboratory electrostimulator (Electro-Science Laboratories, King of Prussia, PA, USA) equipped with platinum electrodes were as follows: the duration of impulse was 3 ms with amplitude of 10 V at 50 Hz during 40 s. The secretion was washed with Milli-Q water (25 mL) into the plastic container with equal volume of methanol. Sample was centrifuged for 15 min at 3000 rpm and filtered through PTF membrane filter (MillexHV, 0.45 μm, Millipore, USA), concentrated at 35 °С with rotary evaporator to 1 mL, lyophilized, and kept at −26 °С.

Mass spectrometric de novo sequencing of the peptides

All LC-MS/MS experiments were conducted using Easy nano-LC 1000 (Thermo Scientific, USA) chromatograph combined to Orbitrap Elite ETD (Thermo Scientific, Germany) mass spectrometer. The laboratory-made chromatographic nano column (75 µm × 150 mm) with stationary phase Aeris 3.6 µm WIDEPORE XB-С18 (Phenomenex, USA) was used. Samples were re-suspended in 1% formic acid and 4% acetonitrile solution in water and injected in 2-μL aliquots. Solution A was 0.1% formic acid in Milli-Q water and solution В was 80% of acetonitrile and 20% of 0.1% formic acid in Milli-Q water. The separation was achieved with the gradient of В from 5 to 60 % in 120 min with eluent current 250 nL/min. Mass spectrometer resolving power in full MS mode was 240000 for m/z 400 and in MS/MS mode—60000 for m/z 400. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) spectra were recorded in automatic mode. The details of experiments were as follows: inlet capillary voltage—1.6 kV, inlet capillary temperature—200 °С, normalized cell energy (NCE) in CID and HCD mode were 28 and 35 correspondingly, activation ETD time—100 ms. Fluoranthene was used in ETD mode.

Fusion Orbitrap mass spectrometer (ThermoFisher Scientific) was used for combined application of ETD and HCD (EThcD experiments) tandem mass spectra. Reversed-phase nano-LC-separation of the peptides was performed with a 50-cm-long EASY spray column (PepMap, C18, 2 μm, 100 Å) at 35 °C. The chromatographic separation was achieved using a gradient solvent system containing (A) water with 2% acetonitrile and 0.1% formic acid and (B) acetonitrile with 2% water and 0.1% formic acid. The gradient was set up as follows: 4% (B) in 7 min, 4–50% (B) in 21 min, 50–80% (B) in 5 min, 80% (B) for 5 min, 95% (B) and 95–4% (B) for 5 min. The flow rate was 300 nL/min. The mass spectrometers were operating in the positive data-dependent acquisition (DDA) mode. A survey mass spectrum was acquired in the range of m/z 300–1700 with a nominal resolution of 120,000 (AGC target of 8.0e5 with a maximum injection time of 50 ms). Precursor ion selection was performed in the “top speed” mode of the charge states from 2 to 7, with the most intense precursor priority and with a minimum intensity of 50,000. Dynamic exclusion duration was disabled. Precursor ion selection for MS/MS was performed for each precursor with EThcD (resolution 15,000, AGC target 5.0e4, activation time 100 ms, maximum injection time 200 ms, collision energy with steps 5%, 10%, 15%, 20%, and 25%, resolution 15,000, AGC target 5.0e4, maximum injection time 200 ms). The sequencing was carried out by manual tandem mass spectra interpretation.

Results and discussion

Peptides identified in the secretion of Slovenian Rana temporaria species are listed in RTS column of Table 1. Peptidome components of Moscow population of Rana temporaria are listed in RTM column of Table 1 [18]. Novel peptides discovered in the secretion of the Slovenian species are marked with *.

Table 1 Peptides identified in the skin secretions of Rana temporaria from the Central Slovenian and Moscow populations
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Central Slovenian population of Rana temporaria is characterized by the same peptide families as the Moscow one (columns RTS and RTM): brevinins 2, brevinins 1, MRT-1, temporins, and bradykinin with numerous analogs and proteolytic copies with N- and C-terminal extension (BRPs).

Disulfide-containing Brevinin 2Т is one of the major secretion components of both Rana temporaria populations (5 in Table 1). It is a potent antibiotic active against gram-positive bacteria Basilus megaterium (LC 0.2 μM) and a wide range of gram-negative bacteria: Enterobacter agglomerans (LC 2.1 μM); Aeromonas hydrophila (LC 30.0 μM); Klebsiella pneumoniae (LC 0.5 μM); Acinetobacter junii (LC 8.5 μM); Escherichia coli (LC 0.5 μM); Yersinia pseudotuberculosis (LC 0.2 μM) [44]. At the early stages of the work with peptide families, quite often there were mistakes in classification of the same peptides. Thus, brevinin 2T was erroneously named ranatuerin 1Т [45]. The mistake was corrected in the paper of D. Barra et al. [44].

Brevinin 2Te is also present in the secretion of both populations. In comparison with brevinin 2T, it has only one substitution (Ser4 for Asp4). Brevinin 2Те was first discovered in the secretion of the Moscow region Rana temporaria species using mass spectrometry [18]. Proteolytic fragment brevinin 2Тf (11–29), found earlier in the Moscow species, was not detected in the secretion of Slovenian Rana temporaria frogs. Its sequence was established by mass spectrometry [18] and slightly differs from the sequence of brevinin 2Тd, isolated from the skin of European frog Rana temporaria [16], possessing two substitutions: Ile18 → Phe18 and Gln20 → Asn20.

Secretion of the Slovenian frogs contains four short disulfide-containing peptides belonging to brevinin 1 family: brevinin 1T, brevinin 1Ta, brevinin 1Tb, and novel brevinin 1Tc. Brevinins 1Т (20 aa) and 1Ta (17 aa) possess antibiotic and hemolytic activities. They were first isolated from the secretion of European frog Rana temporaria [16]. Later, a novel 17-mer brevinin 1Tb (related to brevinin 1Ta) was discovered in the secretion of the Moscow population. Its sequence LVPLFLSKLICFITKKC-OH was established by mass spectrometry using complementary CID and ETD spectra of intact peptide [17]. Identification of Leu4 and Leu9 was achieved taking into account the losses of С3Н7˙ (43,055 Da) from z14 and z9 in ECD mode. Leu6 and Ile10 were defined by the structural analogy with the closely related brevinin 1Та [17]. Brevinin 1Тb is active against Staphylococcus aureus at the concentration 0.015 mg/mL (7.6 μM), being at the same time inactive against Salmonella enterica serovar typhimurium strain [18]. Brevinin 1Т, brevinin 1Тa, and brevinin 1Tb are detected in secretions of both populations (Table 1) and may be considered as biomarkers of Rana temporaria species.

Brevinin 1Tc with disulphide C-terminal loop may be treated as a biomarker of Central Slovenian population of Rana temporaria. The sequence of that novel 17-mer peptide was discovered using complementary HCD and EThcD spectra. Figure 1 illustrates HCD spectrum of triply charged brevinin 1Тс, recorded at NCE 28. The accuracy of measuring of m/z 666.7033+ was 2.3 ppm.

Fig. 1
figure 1

HCD spectrum of m/z 666.7033+ of brevinin 1Tc (NCE 28)

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Methionine in the sequence of intact brevinin 1Тс was identified in the form of methioninesulphoxide (Metox). The abundant ion of m/z 1721.944 corresponds to the neutral loss of 63.998 Da (СH4SO) from the side chain of Metox in ion у15. HCD spectrum contains two series of b/y ions of direct fragmentation of MH33+ ion of brevinin 1Тс and secondary fragmentation of the dominant primary ion у15, forming due to the cleavage of Val-Pro amide bond (b*n in Fig. 1). A pronounced series of у-ions due to direct fragmentation (у7–у16) provides information on the linear portion of brevinin 1Тс, while ion у7 (840.411 Da) corresponds to its 7-member disulfide cycle. The secondary fragmentation of у15 ion results in the opening of S-S cycle by the cleavage of amide bond Lys16-Cys17 remaining S-S bond and forming cystine residue: Cys11Cys17 [36]. HCD spectrum in Fig. 1 demonstrates b*11, b*12, b*13 ions, allowing establishing the following C-terminal sequence of brevinin 1Tc: (CCysFI)TKK. C-terminal loop opening could be more pronounced in conditions of proton deficit [35, 36]. Unfortunately, there was no doubly charged ion in brevinin 1Тс mass spectrum. Figure 2 presents EThcD spectrum of triply protonated brevinin 1Тс. Mass accuracy for m/z 666.7033+ ion was 0.2 ppm. Time activation in ETD was 47.16 ms, normalized HCD activation energy—20.

Fig. 2
figure 2

EThcD spectrum of brevinin 1Tc: m/z 666.7033+, HCD (NCE 20)

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EThcD triggers S-S bond cleavage in intact brevinin 1Tc accompanied by pronounced C-terminal fragmentation. The forming long series of с/z ions allow revealing the sequence of the peptide except two pairs of amino acids (Leu6Ser7) and (Lys8Leu9). Anyway, complementary HCD and EThcD spectra provide the full sequence of disulfide-containing brevinin 1Тс. Two isomeric residues in brevinin 1Тс, although not being confirmed in the experiment, were considered as Leu9 and Ile10 based on the structural analogy with brevinins 1Та and 1Тb. The difference between brevinin 1Tc and 1Tb engages just a single substitution: Leu4→Met4. It is worth specially mentioning that the structure of brevinin 1Тс was established without any preliminary derivatization of S-S bond, i.e., exclusively applying top-down approach and using the benefits of Orbitrap instrument. Certain hitch involves the name of that peptide as the authors [46] erroneously gave that name to the well-known brevinin 1T. Based on the existing nomenclature [7], we decided to correct that mistake and named the novel peptide brevinin 1Тс.

Secretions of both studied populations contain melittin-related peptide (MRP-1), a powerful cytolytic of human red cells (with lethal concentration 0.5 μM). Its structure was first reported by Simmaco in the secretion of the European red frog Rana temporaria [14], while later it was detected in the Moscow population of Rana temporaria [17]. The presence of that peptide in the Slovenian population species renders MRP-1 to be a potential biomarker of Rana temporaria species.

Rana temporaria is a leader among ranid frogs in terms of bradykinin RPPGFSPFR-OH level in their skin secretions [47]. In contrast to vertebrates, producing bradykinin with kallikrein kinin system, amphibian kininogens are synthesized in a unique exocrine apparatus of the skin in the form of inactive precursors—preprobradykinins [48]. Endoproteases release active bradykinin together with the bradykinin-related peptides (BRPs) directly in the amphibian secretion. BRPs affect smooth muscles and participate in the transfer of the pain impulse and in the response to inflammation [49]. Amphibian secretions usually contain a reasonable number of N- and C-terminal extensions of bradykinin and its analogs, proving the presence of numerous copies in their precursors [48]. Bradykinin, various BRPs, and their analogs ([Thr6]Br with an array of С-extended copies and [Asp6]Br) are present in the secretion of the both studied populations of Rana temporaria. Nevertheless, ornitokinin [Thr6, Leu8]Br, an agonist of the ornitokinin receptor, discovered by cloning by Schroeder et al. [50], was not detected in the Slovenian Rana temporaria species. Earlier ornitokinin was discovered in the Moscow population of that specie [17]. According to the modern knowledge, amphibian BRPs are structurally identical to the BRPs of the vertebrates living in vicinity and being potential enemies of the frogs. Amphibian BRPs play a defensive role connecting the receptors of predators notably increasing the pain or triggering vomiting reflex. That identity explains the presence of the conserved structures of amphibian BRPs [48]. It should be emphasized that our observations show that the number of the bradykinin-extended copies in the secretion is defined by the time of interaction of endoproteases with kininogens, i.e., correlate with the level of endoproteases’ deactivation [51].

The main differences in the skin peptidome of Moscow and Central Slovenian Rana temporaria populations involve the temporins family. Slovenian population has 15 temporins, while Moscow one—13, with only 9 being common for both populations. These are temporins A-L [14] and N [18], with the exception of temporins F and K. Temporin M and (Hyp)temporin M are present only in the secretion of Moscow frogs, while Slovenian frogs possess six other (novel) temporins.

Temporin O is a short (13 aa) novel peptide FXGALVNAXRGXX-NH2 (mm 1354.845 Da), with the sequence established using complementary CID, HCD, and EThcD spectra. Temporin О, similarly to the structurally related temporin N, contains one arginine residue, while conserved Pro3 is substituted for Gly3. Figure 3 illustrates corresponding CID, HCD, and EThcD spectra of doubly charged ions of temporin O of m/z 678.430 (accuracy 3.1 ppm). One can see that HCD spectrum is notably more informative than the lower energy CID spectrum. It demonstrates more ions peaks of higher intensity, including low mass species and even a peak of the protonated molecule. Combination of CID and HCD spectra provides the whole peptide sequence, including N-terminal residues (bond cleavage Phe1-Leu2, y12—m/z 1208.784).

Fig. 3
figure 3

Tandem mass spectra of the 678.4302+ ion of temporin O: (a) CID (NCE 35); (b) HCD (NCE 28); (c) EThcD (ETD 106.1 ms); HCD (NCE 5.00)

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Identification of isomeric Leu/Ile was achieved in EThcD mode, involving application of HCD to the whole array of the primary ions formed in ETD mode. It is worth mentioning that the collision energy in HCD may be varied. EThcD spectrum of temporin O is more informative than the ETD one (not presented). It is not surprising as the formed in ETD radical cations receive additional excitation at the HCD stage, which promotes fragmentation. Leu5 was identified by the characteristic loss of С3H7˙ 43.055 Da from z9 (m/z 950.616) primary fragment ion.

Temporin P (1319.854 Da) is another 13-mer peptide, characteristic for the Central Slovenian population, with conserved Pro3 and the absence of basic amino acids in the sequence. Due to that peculiarity, even combination of CID and HCD spectra, including secondary fragmentation pathways, was not very efficient. Nevertheless, EThcD spectrum (Fig. 4) allowed elucidating the full sequence.

Fig. 4
figure 4

EThcD spectrum of m/z 660.93422+ ion of temporin Р: ETD 106.10 ms; HCD (NCE 10.00), mass accuracy 0.3 ppm

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Ion z12 of m/z 1190.752 (accuracy 0.6 ppm) corresponds to peptide bond cleavage between N-terminal Leu1–Val2. Thus, full sequence was obtained with complementary CID, HCD, and EThcD spectra. The isomeric Leu/Ile residues in temporin Р were not differentiated, as it does not contain even a single basic aa. As a result, the intensities of the primary z-ions are very low. Leucines were mentioned in the sequence by analogy with other temporins.

Temporin Q (1700.050 Da) is one of three 16-mer peptides of that family in the secretion of the Slovenian Rana temporaria species. Moreover, these three peptides contain two basic residues (temporin Q—Lys and Arg, temporins R and S—two Arg). Figure 5a and b demonstrate CID and HCD spectra of doubly charged temporin Q of m/z 851.0312+ (accuracy 1.9 ppm).

Fig. 5
figure 5

Tandem mass spectra of m/z 851.0312+ ion of temporin Q: (a) CID spectrum (NCE 35); (b) HCD spectrum (NCE 28); (с) EThcD spectrum of m/z 567.6913+ ion (accuracy 0.5 ppm) of temporin Q: ETD 47.16 ms; HCD 20 (NCE). *Secondary fragment ions of y14 precursor

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The spectra also illustrate schematically the obtained structural information. Fragmentation of temporin Q remains similar at higher collision energy. One can see the same ratio of peaks of the primary and secondary fragmentation ions (Fig. 5а, b). HCD spectrum shows also protonated molecule and extended b-series prolonged to the low masses. Complementary CID and HCD spectra allowed elucidating full temporin Q sequence, including N-terminal residue. Identification of Leu1, Leu12, and Leu13 was carried out using EThcD spectrum [42, 43] illustrated in Fig. 5c by the corresponding losses of isopropyl radicals from the following primary ions: z16 (m/z 1684.032), z5 (m/z 601.382), and z4 (m/z 488.299).

Temporin R (1964.072 Da) as well as temporin S (1728.056 Da) differs from the other known temporins of Rana temporaria species. These are 16-mer amidated peptides with conserved Pro3 and two arginine residues in the molecule. Figure 6a illustrates CID spectrum of m/z 848.0432+ ion (accuracy 0.7 ppm) of temporin R.

Fig. 6
figure 6

(a) CID spectrum of m/z 848.0432+ ion of temporin R: 0.7 ppm; NCE 35; (b) EThcD spectrum of m/z 565.6983+ ion (accuracy 0.5 ppm) of temporin R: ETD 47.16 ms; HCD 25 NCE

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Despite the fact that fragmentation of that doubly charged ion proceeds at conditions of proton deficit due to the presence of two arginine residues (Arg7, Arg15), the spectrum shows the peaks of primary and secondary (*) ions. The latter arise due to the fragmentation of abundant y14 ion (N-Pro3 cleavage). Series of b/y ions of direct fragmentation and the series of secondary b*-ions provide full sequence of temporin R, except interposition of (LeuVal) pair. Ions of y-series of temporin R easily lose NH3 molecule (у010, у011, у012, у013, у014). Some of the forming ions lose another NH3 molecule due to the fragmentation in the side chains of Arg residues (marked у0´n): у0´3, y0´5, y0´7, y0´11, y0´13, y0´14, y0´15. NH3 loss from the side chain of Arg15 takes place also from b16 ions (b016, m/z 1660.042). EThcD spectrum helps confirming the sequence of temporin R and identifying three leucine residues (Leu9, Leu12, and Leu13) by the neutral loss of isopropyl radicals С3Н7˙ (43.055 Da) from z8, z5, and z4 ions (m/z 794.526; 567.399, and 454.315, correspondingly) (Fig. 6b).

Therefore, de novo sequencing of temporin R ХVPFХGRTLGGLLARХ-NH2 including identification of Leu9, Leu12, and Leu13 was done using complementary CID(2+) and EThcD(3+) spectra.

Temporin S (1728.056 Da) differs from temporin R by only one substitution X16–Phe16: LVPFХGRTXGGLLARF-NH2. Its sequence was obtained similarly to temporin R by the complementary (CID(2+) + EThcD(3+)) spectra. Leu1, Leu12, and Leu13 were identified in EThcD spectrum of triply charged ion of m/z 577.0293+ (accuracy 1.7 ppm).

The sequences of 19 temporins (Table 2) belonging to the genus Rana temporaria are known by now. The sequences are rather variable both in terms of the chain length and substitution of residues. Nevertheless, certain conservancy of the peptides primary structure is observed inside Rana temporaria peptidome. Novel temporins discovered in the present study are marked with (*) in Table 2. The number of possible substitutions relatively temporin A in that particular position of the backbone is marked with (**) at the bottom of Table 2. Unassigned isomeric Leu/Ile residues are marked as X.

Table 2 Temporins from the skin secretion of Rana temporaria
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Temporins belonging to Rana temporaria species may be divided into two subfamilies: short (10–13 аа) and long (16 аа) ones. All of them are amidated. Temporins H and K lack three residues in positions 4–6 of the sequences, i.e., have only 10 residues in the chain. Two positions in Rana temporaria temporins are conserved: only Leu is known to occupy positions 9 and 13 so far. Although it was not reliably proved for temporins M–Q, S, and T, it is highly likely that Leu rather than isomeric Ile occupies those positions. Only two substitutions are possible in five positions of the short (10–13 аа) temporins (1, 6, 7, 11, and 12). Thus, Phe1 of temporin А may be substituted only for Leu1 or Val1; Gly6 may be substituted for Ser6 or Val6; Arg7 may be substituted for Asn7 or Lys7; Gly11 may be substituted for Ser11 or Arg11; аnd Ile12 may be substituted only for Leu12 or Val12. Fourteen out of 19 temporins (Table 2) have Pro3; however, it may be substituted for Hyp3, Gln3, or Gly3. The most variable in the short temporins (10–13 аа) of Rana temporaria species are positions 4, 8, and 10, where five possible residues were identified. Speaking about conserved sites of the long temporins Q, P, and S, it should be mentioned that substitutions in their backbone occur only in positions 5, 7, and 16. Table 2 may be very helpful for the de novo sequencing of novel temporins of Rana temporaria species, as that family appeared to be the most variable in their composition and novel sequences may be discovered in other populations of that common frog.

Biological activities of brevinin 1Тс and six novel temporins (O, P, Q, R, S, T) may be estimated using 2D mass mapping [52]. Peptides of the same family usually demonstrate similar type of bioactivity and occupy neighboring positions in 2D map with normalized mass defect (NMD) and normalized isotopic shift (NIS) as coordinates. Figure 7 presents such 2D map, demonstrating novel peptides marked with (*) and the known peptides of Rana temporaria, including brevinins 1 and temporins with the studied bioactivity. Three novel short temporins (O*, P*, and T*) are situated very close to temporins А and B. The latter ones are the most studied so far, while their bactericidal, fungicidal, and hemolytic activities are reported [14].

Fig. 7
figure 7

2D mass map of novel peptides identified in the skin secretion of the Slovenian frogs Rana temporaria

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Both temporins A and B are active against various gram-positive bacteria and fungi, being significantly less active against gram-negative species. Their hemolytic activity is low. Three long (16 аа) temporins (Q*, R*, S*), with two basic residues in the sequence, are shifted along NMD axis to higher values similarly to temporin L. The latter, also having two basic residues in the backbone, demonstrates wide spectrum of activities against gram-positive and gram-negative bacteria, yeast, human erythrocytes, and tumor cells [53].

Temporins without basic residues, i.e., novel temporin P, are usually not bioactive. Nevertheless, related temporin 1Od from Rana ornativentris inhibits the growth of S. aureus [54]. Novel brevinin 1Тс* occupies somehow distant position from other brevinins: brevinin 1Т and its homologues, brevinins 1Тa and 1Tb, possessing bactericidal activity [16, 17].

The study of correlations structure/activity for temporins allows proposing several requirements to make them bioactive. These are high hydrophobicity (up to 70% аа), ability to form α-helix, and the presence of basic amino acids in the sequence. Table 3 summarizes the values of hydrophilicity, α-helicity, and net charges at pH 7 of the Rana temporaria temporins, calculated using the Proteus [56] and Bachem online software [55].

Table 3 Physicochemical and structural properties of the temporins from Rana temporaria
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All Rana temporaria temporines are not 100% α-helices as certain portions of their sequence form random coils. Only two-thirds of the sequences of the most active temporins (A and B) demonstrate α-helical form. Novel temporins О* and T* having similarity to temporins А and В values of hydrophilicity and net charge demonstrate even higher value of α-helicity: 77% of the sequence vs 62 and 69% for temporins А and В correspondingly. Speaking about novel triply charged temporins Q*, R*, and S*, one should mention that the extension of their sequences by 3 amino acids slightly decreased their hydrophilicity and notably decreased their α-helicity (up to 0 for temporin Q*). The present knowledge is still far away from the rationalization of the role of each peptide in the amphibian secretion. Maybe various homologue peptides protect frogs from numerous possible pathogenic invasions and opportunistic infections [20].

The confirmed chemotactic effect of temporin A involving cell surface receptors of human phagocytes [25] may be treated as a bridge between innate and adaptive immune systems. One could suppose that adaptation of the Central Slovenian population of Rana temporaria to the living conditions involved temporins. The array of temporins in their skin peptidome is alternated due to the developed formation of six novel peptides of that family which is the most promising from the point of view of drug development.

Application of automatic sequencing engines

Numerous previous searches with the available automatic sequencing engines were not successful when they dealt with de novo sequencing of natural frog peptides [36]. However, the last attempt to apply PEAKS Studio for the sequencing of Cuban banana frog [39] was quite promising. Thus, PEAKS was applied to the raw data obtained in CID and HCD modes. The results were rather interesting as the sequences of numerous bradykinins, with Arg1 and Arg9, in the backbone were established with 100% success. Full sequences of seven temporins, including three novel ones, were also confirmed by the PEAKS Studio algorithm. Three temporins were sequenced by that engine with minor mistakes, while the proposed sequences of two temporins were completely wrong, and five species were not detected by the program at all. On the other hand, the automatic sequencing allowed discovering another novel temporin missed during manual approach (temporin T—FХGALVNAХTRVХ-NH2). The results were much worse with brevinins. In some cases, the linear portion of the sequence was established; however, the C-terminal cycle was never determined, although manual interpretation of both CID and HCD spectra provided the corresponding sequence information due to S-S bond cleavage. MRP-1 (22 aa) was also missed by the automatic sequencing algorithm. Anyway, automatic sequencing engines will be used in our future studies since they can provide additional information to manual sequencing and may be very useful in getting the correct sequence of natural peptides.

Conclusions

Skin peptidomes of common frog species with extended habitats may differ in various populations. The Central Slovenian population of brown frog Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be considered as biomarkers of that particular population.

Complementary CID, HCD, and EThcD tandem mass spectra obtained with high resolution and high mass accuracy are the advantages of modern Orbitrap mass spectrometers allowing for reliable de novo sequencing of natural peptides with top-down approach, including the differentiation of the isomeric pair Leu/Ile. EThcD mode may also cleave S-S bonds in the disulfide-containing peptides, eliminating the preliminary sample preparation stage with obtaining of S-derivatives.

Important information on the primary structure of Rana temporaria peptides complementary to manual sequencing was obtained by the PEAKS Studio sequencing algorithm, proving that automatic engines may be quite successful in de novo sequencing of natural peptides.