Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides Tatiana Y. Samgina, Egor A. Vorontsov, Vladimir A. Gorshkov, Konstantin A. Artemenko, Roman A. Zubarev, Jimmy A. Ytterberg, et al. Journal of The American Society for Mass Spectrometry The official journal of The American Society for Mass Spectrometry ISSN 1044-0305 Volume 24 Number 7 J. Am. Soc. Mass Spectrom. (2013) 24:1037-1044 DOI 10.1007/s13361-013-0632-y

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B American Society for Mass Spectrometry, 2013

J. Am. Soc. Mass Spectrom. (2013) 24:1037Y1044 DOI: 10.1007/s13361-013-0632-y

RESEARCH ARTICLE

Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides Tatiana Y. Samgina,1 Egor A. Vorontsov,1 Vladimir A. Gorshkov,1,4 Konstantin A. Artemenko,2 Roman A. Zubarev,3 Jimmy A. Ytterberg,3 Albert T. Lebedev1 1

Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russian Federation Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden 3 Department of Medicinal Biochemistry and Biophysics, Division of Molecular Biometry, Karolinska Institutet, Stockholm, Sweden 4 Present Address: Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, Bldg. 16, 35392 Giessen, Germany 2

Abstract. Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S–S bonds is often used in “bottom up” sequencing approach. However, lowenergy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the “hidden” C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S–S bond rupture does not occur in this case. Key words: Collision-induced dissociation, Non-tryptic natural peptides, Disulfide cycle, Peptide fragmentation, Peptide sequencing, Lysine Received: 18 December 2012/Revised: 27 March 2013/Accepted: 27 March 2013/Published online: 30 April 2013

Introduction

D

isulfide bond formation is one of the most widespread post-translational modifications (PTM) of peptides and proteins. Disulfide bonds play an important role in biological functioning of proteins. There are two types of disulfide bonds: intrachain (connecting cysteine residues in one peptide chain) and interchain (connecting separate peptide chains in a protein). In both cases, S–S bonds are formed by side chains of cysteine residues. Intrachain disulfide group represents a certain problem for de novo sequencing of peptides with mass spectrometry, as part of the sequence remains hidden. S–S bond reduction followed by thiol groups’ derivatization is often used in “bottom up” sequencing approach. Iodoacetamide is the most popular derivatization reagent so Correspondence to: Albert T. Lebedev; e-mail: [email protected]

far. It may be successfully applied in the studies of various substrates, including long natural non-tryptic peptides [1]. Besides iodoacetamide, a wide variety of other reagents have been applied for cysteine derivatization [2, 3]. Moreover, some matrixes in MALDI may trigger S–S bond reduction during sample application on the target plate [4] or directly during mass spectrometry experiment [5]. In “top-down” approach, different instrumental features, first of all, various methods of fragmentation initiation are applied for S–S bond cleavage. Interchain disulfide bonds break up easier than intrachain ones. CID [6], MALDI-postsource decay (PSD) [7], electron capture dissociation (ECD) [8], ultraviolet photodissociation (UVPD) [9], or negative ion mass spectrometry [10, 11] have been used to achieve cleavages of interchain S–S bonds and to establish sequences of separated individual peptide chains at the next stage of mass spectrometry experiment.

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Peptides with intramolecular disulfide bonds have been successfully investigated using ECD [8, 12, 13] and electron transfer dissociation (ETD) [14–18]. These techniques provided positive results for small (less than 15 amino acid residues), usually tryptic peptides. Negative ion mass spectrometry demonstrated its efficiency for sequence determination inside S–S loops for various types of peptides, including natural non-tryptic ones [19]. Low-energy collision-induced S–S-cycle disruption depends on charge state of precursor ion [20]. Monoprotonated insulin fragments in CID mode with predominant rupture of S–S bond. Fragmentation of doubly and triply protonated insulin species results in the cleavage of both S–S and NH– CO bonds. Finally, ions (М+4Н)4+ and (М+5Н)5+ yield in CID exclusively ions due to amide bonds cleavages. Therefore, protonated peptide bond cleaves easier than S–S bond, but absence of a mobile proton in low charge state ions facilitates disulfide bond cleavage. The latter process may involve charge remote fragmentation, especially in large protein molecules [20]. These results were supported by experiments and calculations conducted for small model peptides with S–S bond [21]. Presence of a mobile proton in the protonated molecules of disulfide-containing peptides is considered to be the most important factor directing amide bonds cleavages at low-energy collisions. Most cleavage processes involving disulfide bond fragment (S–S or C–S bonds) lie outside the band of energy required to cleave the amide bond in a peptide, with the exception of the salt bridge mechanism to cleave the C–S bond. When fragmentation occurs in the absence of a mobile proton (e.g., in singly charged (GCR)2 molecule) C–S bond cleavage is readily observed [21]. However, Gorman et al. [22] mentioned that disulfide bond cleavages in CID are rarely observed for larger tryptic peptides, suggesting that disulfide bond cleavage processes have higher relative energy barriers compared with amide bond cleavages. Mormann et al. [23] demonstrated that fragmentation inside S–S loop at low-energy CID is preceded by protoninduced asymmetric cleavage of the disulfide bridge, resulting in formation of dehydroalanine (dhA) and Ssulfanylcysteine residues. This cleavage leads to b-ions, shifted for −34 mass units and y-ions shifted for +32 mass units. These ions were proposed to be called α- and β-ions [23]. We consider this name dubious as these terms are widely used in the nomenclature of deprotonated peptides’ fragment ions [24, 25]. Proline inside S–S loop considerably facilitates cleavages of the neighboring amide bonds both in ESI CID [23, 26] and МАLDI-PSD modes [27]. Thakur and Balaram studied MALDI-PSD fragmentation of 8- 9-member peptides (components of Conus venoms) containing C-terminal S–S loops. They showed that in these conditions, the cleavage of amide bonds at N-terminus of Pro or hydroxyPro (Hyp) situated inside S–S loop was followed by disulfide bond rupture [27]. These consecutive processes resulted in the formation of asymmetric acyclic ions, including dehydroalanine and R-

S-SH fragment. The formed ions fragmented by a number of pathways under MALDI conditions. Resulting MS/MS spectra contained many signals due to facilitated cleavages of weak N-terminal bonds of Pro and Hyp followed by secondary fragmentation processes involving amide bonds ruptures accompanied by sulfur atom and ammonia losses. In the present paper, the results on low-energy CID fragmentation of eleven intact peptides from five frog species belonging to genus Rana: R. temporaria, R. esculenta, R. arvalis, R. lessonae, and R. ridibunda are reported. Spectra were obtained during routine analysis of frog skin secretions, while the sequence was reported earlier [1]. Since the spectra contained a lot of intensive peaks, which do not belong to b/y-series, we decided to clarify the nature and mechanisms of formation of the corresponding ions. The obtained results reported recently at the 19th IMSC in Kyoto [28] appeared useful for the estimation of applicability of direct low energy CID fragmentation in conditions of routine proteomic analysis for the de novo sequencing inside C-terminal disulfide cycles (Rana box) of intact natural non-tryptic peptides (15–35 аа). The data allowed clarifying the mechanisms of alternative directions of fragmentation of these peptides (i.e., does asymmetric cleavage of S–S bond precede amide bonds cleavages inside S–S cycle or vice versa?) The data are reported, taking into account the charge state of the precursor ion.

Experimental General Methanol and acetonitrile were of HPLC grade (Acros, Geel, Belgium). Trifluoroacetic acid (TFA; 98 %) and formic acid (98 %) were also purchased from Acros. Water was distilled in-house.

Skin Secretions Specimens of ranid species of Rana temporaria, Rana esculenta, Rana arvalis, Rana lessonae, and Rana ridibunda were caught near Zvenigorod in the Moscow region (Russia). Animals were maintained in captivity under conditions close to natural ones. Secretion from the skin glands was obtained by mild electrical stimulation [29]. The procedure details have been described previously [30]. Aqueous methanolic solutions of skin secretion were concentrated at 35 ºС using rotary evaporator to a volume of ca. 1 mL and lyophilized. Lyophilized samples were kept in a freezer at −26 ºС.

HPLC Purification of the Skin Secretions The procedure details have been described previously [30– 33]. HPLC system from ThermoSeparation Products,

Author's personal copy T. Y. Samgina et al.: CID Cleavages Inside Disulfide Peptide Cycle

equipped with binary gradient pumps ThermoSystem P2000 (Piscataway, NJ, USA) and a reversed-phase analytical column C18 (Reprosil-Pur, 5 μm, 100 Å, 150×4 mm; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) was used. The single injection volume was 20 μL to improve separation. Absorption was measured at 214 nm (UV detector Spectra System UV3000). The fractions with retention time 10.0–60.0 min were combined, concentrated at 35 ºС using rotary evaporator to a volume of 1 mL, and lyophilized. Dry samples were stored at −26 °C.

Mass Spectrometric Sequencing of the Peptides All experiments were performed on a hybrid ion trap-Fourier transform ion cyclotron resonance (FT ICR) instrument (LTQ FT Ultra; Thermo Fisher Scientific, Bremen, Germany), equipped with 7 Tesla superconducting magnet and coupled to nano-ESI ion source (Proxeon Biosystems, Odense, Denmark). The samples were desolved in acetonitrile/water/formic acid mixture (1:1:0.002 vol/vol/vol). Separation of peptides was achieved by an Agilent 1100 nanoflow system equipped with a 15-cm fused silica emitter (Proxeon Biosystems) packed in-house with a Reprosil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, HPLC), and coupled online to the mass spectrometer. The following solvents were used: A −99.9 % HPLC grade water with 0.1 % formic acid, B −90 % acetonitrile with 0.1 % formic acid. The peptide separation was done using linear gradient from 4 % to 80 % (70 min) solvent B at 200 nL min–1. Mass spectrometer was operated in positive ion mode and during each run it was automatically switched between a high resolution (resolving power 100,000 FWHM at m/z 524) survey mass spectrum in the FT-ICR cell and consecutive highresolved (25 000 FWHM at m/z 524) ECD and CID spectra of two most abundant ions in FTMS. CID was performed with helium as a collision gas (pressure 0.1 Pa). Normalized collision energy was set to 30 %. Interpretation of the spectra was performed manually.

Results and Discussion Direct fragmentation of 11 disulfide-containing peptides (Table 1) was studied in low-energy CID conditions. Spectra were registered in routine analysis of frogs’ skin secretions with nanoHPLC-ESI-MS system. The sequence of these peptides was established and reported earlier [30–33]. All the studied peptides (15–35 residues) possess a common feature: С-terminal 6–7 member S–S loop. Since all these peptides contain several basic amino acid residues in their backbones, ESI conditions promoted formation of multiprotonated species (in contrast to tryptic peptides that are usually doubly charged). The studied peptides belong to four families: brevinins-1 and 2, ranatuerins-2 and esculentins-2 (Table 1), differing by

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the size of S–S loop and, what is more important, by the number and position of basic Lys residues: brevinins 1 brevinins 2 esculentins 2 ranatuerins 2

Cys Cys Cys Cys

X X X Lys Lys X X X Lys X X X Lys X X X

Lys Cys X Cys X Cys Cys

Since basic residues with high proton affinity in all the studied peptides are neighboring cysteine residues forming S–S bond, it was possible to expect C–S bond cleavage via salt-bridge mechanism [21] in the absence of mobile proton. Nevertheless, thorough search for (b– 34) and (у+32) ion series, forming by asymmetric cleavage of S–S bond [23], in CID spectra has not revealed such ions for any of 11 disulfide peptides. However, intensive signals of m/z 1314.82, 1467.80, 1554.84, 1667.93, 1768.97, and 1897.07 were recorded in CID spectrum of doubly charged brevinin1Та (Figure 1). These ions could not be rationalized by any of the accepted fragmentation schemes proposed for low energy CID mode. Exact mass differences between these signals correspond to consecutive losses of Lys, Lys, Thr, Leu/Ile, and Ser from [M+2H]2+ ion of brevinin-1Та. This set represents exact amino acid sequence inside “Rana box” of brevinin-1Та: SITKK (Figure 1). The ions might appear as a result of amide bond Lys16–Cys17 cleavage, while S–S bond remains intact (see Scheme 1 arrow a). In another words, doubly protonated 17-member brevinin-1Ta with C-terminal loop FITLLRKFICSITKKC-OH isomerizes into acyclic 16member ion А containing cystine with the kept water molecule (CC) in position 11: FITLLLRKFICCSITKK. Ion A being isomeric to the initial protonated peptide molecule fragments both from N- or C-termini, its C-terminal fragmentation results in the formation of several b*-type ions containing cystine (b*12-b*16) due to amide bonds’ cleavages at the C-terminal inside the former cysteine cycle (see Scheme 1). Therefore, based on the character of the formation and their intrinsic structure b*-ions represent bions. However, formally (on the basis of their composition) they are y-ions as cystine residue in position 11 contains water molecule. The C–S bond rupture in cystine takes place when b*11-ions of m/z 1467.80 eliminate S-sulfanylcysteine (152.98 mass units) accompanied by hydrogen migration and formation of m/z 1314.82 b11 ion with dhAla at its Cterminus (see Scheme 1). Scheme 1 represents the proposed processes of fragmentation of protonated molecule of brevinin-1Та in conditions of low-energy CID. Ion b10 may be formed as a result of usual fragmentation of amide bond Ile10–Cys11, as well as due to the cleavage of SS-loop according to Scheme 1. Cystine-containing ions b* (b*11–15) are formed because of C-terminal fragmentation of acyclic ion A (shown clockwise in Scheme 1) from one another similarly to the known formation of lower b-ions from the higher b-ions [34]. All of them can also arise

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Table 1. Sequence Coverage (%) of Intact Ranid Peptides with S-S Loops (ESI LE CID Spectra) №

MWexp

1

2024.14

Brevinin-1Ta

2

1963.13

Brevinin-1 Tb

3

2195.28

Brevinin-1 T

4

1809.93

Brevinin-1AVa

5

1873.08

Brevinin-1AVb

6 7

2607.45 3618.97

Brevinin-1LE Esculentin-2LE-(3–37)

8

3011.61

Brevinin-2Rd

9

2989.61

Brevinin-2Ra

10

1748.98

Ranatuerin-2Ra

11

1939.02

Ranatuerin-2R

Peptide name

z

Sequences

%

3 2 Σ 4 3 2 Σ 4 3 2 Σ 3 2 Σ 3 2 Σ 3 6 5 4 Σ 5 4 3 Σ 5 4 3 Σ 3 2 Σ 3 2 Σ

FITLL(LR)KFI(CSITK)KC-OH (FITLL)LRKFICSITKKC-OH FITLLLRKFICSITKKC-OH LVPLF(SKLICFITKKC)-OH LVPLFLSKLI(CFITK)KC-OH LVPLFLSKLI(CF)ITKKC-OH LVPLFLSKLI(CF)ITKKC-OH VNPIILGVL(PK)FV(CLITKKC)-OH VNPIILGVL(PK)FV(CLI)TKKC-OH (VN)(PI)ILGVLPKFV(CLI)TKKC-OH VNPIILGVLPKFV(CLI)TKKC-OH FLPLLAASF(ACTVTKKC)-OH FLPLLAASFACTVTKKC-OH FLPLLAASFACTVTKKC-OH FVPLLVSKLV(CVV)(TK)KC-OH (FV)PLLVSKLVCVVTKKC-OH FVPLLVSKLVCVVTKKC-OH FFPAFLKVAKVV(PS)IL(CSITKKC)-OH (FS)(LVKGVAKLAGK)(TLAK)E(GGKFGL)DLIA(CK)(ITNQC)-OH (FS)LV(KGVAK)LA(GK)(TL)AKE(GGKF)(GL)DLIA(CK)I(TN)QC-OH (FS)LVK(GV)AKLAGKTLAKEGGKF(GL)DLIACKITNQC-OH (FS)LVK(GV)AKLAGKTLAKEGGKF(GL)DLIACKITNQC-OH (GI)L(DSLK)NLA(KN)AAQILL(NK)(AS)(CKLSGQC)-OH (GI)LD(SL)KNLAKNAAQILLNKAS(CKLSGQC)-OH (GI)LDSLK(NL)AKN(AA)QILLNKAS(CKLSGQC)-OH (GI)LDSLKNLAKNAAQILLNKAS(CKLSGQC)-OH (GI)LD(SL)KNFAKDAAGILLKKAS(CKL)(SGQC)-OH (GI)LDSLKNFAKDAA(GI)LLKKA(SCKLSGQC)-OH (GI)LDSLKNFA(KD)AAGILLKKASC(KLSGQC)-OH (GI)LDSLKNFAKDAAGILLKKASC(KL)(SGQC)-OH KLLLNPKFR(CKAAFC)-OH KLLLNPKFRC(KAAFC)-OH KLLLNPKFRC(KAAFC)-OH AVNIP(KFKV)K(FRCKAAFC)-OH AVNIPKFKVKFRC(KAAFC)-OH AVNIPKFKVKFRC(KAAFC)-OH

58.8 70.6 100.0 35.3 64.7 88.8 88.2 55.0 75.0 65.0 85.0 52.9 100.0 100.0 70.6 88.2 100.0 58.3 13.5 35.1 82.9 82.9 34.5 62.1 55.2 69.0 62.1 58.6 65.5 72.4 60.0 66.7 66.7 33.3 72.2 72.2

z0precursor ion charge state; Σ0overall coverage taking into account all charge states of the peptide; %0sequence coverage; Lys residues (K) are shown in bold

directly from acyclic ion А eliminating corresponding particles containing several amino acid residues. Ион b*11 loosing S-sulfanylcysteine and dhAla transforms into normal ion b10. The alternative direct formation of the latter is shown by arrow b (Scheme 1). Low-energy CID conditions favor cleavages of protonated amide bonds in disulfide containing peptides [34], as this process requires the lowest energy in comparison to the competitive processes [21, 22]. At higher collision energies, the selectivity is not so pronounced. However, the influence of the charge state (proton deficit) is more important, as the direction of LE CID fragmentation strongly depends on the degree of protonation of the peptide molecule [20]. Brevinin-1Та contains Arg outside S–S loop. Basic arginine residue has the highest proton affinity among all amino acids, thus it firmly binds protons that become immobile [35]. In case of doubly protonated brevinin-1Ta, the second proton can hardly be a mobile one as the molecule contains three lysine residues, also possessing very high proton affinity. As a result, the second proton is attached to one of these lysines and fragmentation in CID conditions mainly involves charge site initiation in the

vicinity of the basic amino acid residues. Since two Сterminal Lys are situated inside “Rana box,” the cleavage of Lys–Cys amide bond at C-terminus also becomes rather favorable. Formation of acyclic b*16-type ion А isomeric to cyclic protonated molecule rationalizes the appearance of further b*11–15-ions. The corresponding processes reveal the sequence inside “Rana box,” although they do not require S– S bond cleavage. Table 1 summarizes results of manual sequencing of 11 intact natural peptides, taking into account the mentioned fragmentation inside disulfide cycle. The Table represents the sequence coverage depending on the degree of protonation (z) of the precursor. Undetermined regions of the peptide sequences are enclosed in brackets. The table also contains the overall sequence coverage calculated by taking into account the spectra of all the charge states of the precursor ions of the peptides studied. Table 1 demonstrates that five brevinins-1 out of six undergo fragmentation depicted in Scheme 1. The best sequencing results inside “Rana box” are usually obtained for the least protonated species (+2). Brevinin-1LE represents an exception, since its doubly protonated precursor ion was not observed. The reason may involve the presence of

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Figure 1. CID spectrum of brevinin-1Ta ([M+2H]2+ precursor ion, after charge deconvolution)

one more Lys in its molecule. Thus, formation of higher charge states becomes preferable. Since all the studied peptides contain several basic residues in their backbone, all the precursor ions (often including (М+ 4Н)4+ and (М+3Н)3+) are proton deficient (i.е., the number of attached protons is lower than the number of the basic residues in the peptide) and should not have a mobile proton. Nevertheless, the discussed fragmentation inside disulfide cycle becomes less pronounced with each new proton, losing competition to the classic backbone cleavages outside the cycle. At the same time, the information obtained from the corresponding spectra was quite useful and complementary (especially for the full sequence coverage) to that provided by CID spectra of (М+2Н)2+ precursor ions.

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The discovered fragmentation inside "Rana box" can take place not only in the whole protonated peptide molecule, but also in the most abundant y-ions. Peptides containing Pro demonstrate intensive y-ions because of the cleavages of amide bond at N-terminus of this residue. Figure 2 represents CID spectrum of proline-containing peptide brevinin1AVb. It demonstrates three series of b-ions: direct fragmentation of the protonated molecule (bn), fragmentation of the isomeric acyclic protonated molecule (bn*), and secondary fragmentation of the dominant ion у15 (m/z 1627.96), formed because of the amide bond cleavage at N-terminus of Pro3 (y15bn*). Actually, it is difficult to say either S–S cycle is cleaved after у15 ion is formed, or amide bond at Pro3 cleaves in the corresponding ions of bn* series. Ions y15b13*–y15b10* (m/z 1499.86, 1371.79, 1270.74, and 1171.67) correspond to the following losses from у15ion (PLLVSKLVCCVVTKK): –Lys, –Lys, –Thr, and –Val. The same losses from the protonated molecule of brevinin-1AVb are less pronounced: (b15* – 1746.00, b13* – 1516.85, b11* – 1165.74). These ions correspond to the elimination of Lys, –(LysThr), – (ValVal+ Ssulfanylcysteine). Six “normal” ions b5–b10 may appear from both intact and linear protonated molecule of brevinin-1AVb: FVPLLVSKLVCVVTKKC-OH. It is possible to elucidate complete sequences of four out of six (nos. 1, 2, 4, 5 in Table 1) brevinins-1 exclusively on the basis of low-energy CID data and sequence similarities inside peptide families (i.e., without any chemical derivatization or even cleavage of S–S bonds). Definitely, the presence of the additional C-terminal Lys favors proton attachment at this part of the molecule and contributes to the

Scheme 1. ESI low energy CID fragmentation pathways intra SS loop of brevinin-1Ta

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Figure 2. CID spectrum of MH22+ ion of brevinin-1AVb. CC 0cystine residue; y15bn*0secondary fragment ions of cystine containing y15-ion (m/z 1627.96); bn*0ions from fragmentation of the isomeric linear protonated molecule of brevinin-1AVb

fragmentation process inside S–S loop. This process is especially efficient when we have a proton-deficient precursor ion and the probability of competitive reactions induced by mobile proton is significantly reduced. Peptides 7–11 do not contain two C-terminal Lys in the chain. As a result, fragmentation inside “Rana box” is not so

pronounced and sequence coverage is lower. Nevertheless, ESI CID spectrum of (М+4Н)4+ ion of esculentin-2LE-(3– 37) with 6 Lys in the backbone demonstrated quite intensive fragmentation inside “Rana box” (Figure 3). Peaks of ions of m/z 3491.92, 3377.88, 3276.83, 3163.75, 3035.66, and 2813.66 are clearly seen in the zoomed region

Figure 3. Charge-deconvoluted ESI CID spectrum of (М+4Н)4+ ion of esculentin-2LE-(3–37): (а) the whole spectrum; (b) zoomed region in the range of m/z 2800–3500; b*0ions formed by opening of S-S loop and formation of isomeric precursor ion; CC 0cystine residue

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of the spectrum (m/z 2800–3500, Figure 3b). These ions correspond to the sequential losses of Gln, Asn, Thr, Ile, Lys, and cystine from (М+4Н)4+ ion of esculentin-2LE-(3– 37). This is the reverse amino acid sequence inside Cterminal cycle of this peptide: KITNQ. Being proteolytic fragment of the reported earlier [32] esculentin-2-LE, this peptide was reported in the present study for the first time. Liu et al. [36] reported migration of oxygen atom from Gln36 to Asp35 working with closely related esculentins2JDa and 2JDb. This rearrangement was not observed in the case of esculentin-2LE-(3–37) with the same structure of “Rana box”: CKITNQC. Accurate mass measurements with FT ICR mass spectrometer demonstrated only the loss of Gln34 residue followed by the loss of Asn33 residue through bx →bx-1 process during fragmentation of (М+4Н)4+ ion of esculentin-2LE-(3–37) (Figure 3). High abundance of the ions forming because of cleavages inside “Rana box” for the peptide without two C-terminal Lys may be rationalized by the presence of acidic Asp25 in the backbone. As esculentin2LE-(3–37) contains six basic Lys residues in its structure even (М+4Н)4+ ion of this peptide does not contain any mobile proton for the realization of efficient CID backbone amide bonds cleavages. Therefore, in conditions of proton deficit Asp25 residue contributes to the fragmentation, donating its side chain acidic proton [37]. The same rationalization may be proposed for the fragmentation of esculentins, 2JDa and 2JDb, with Glu27 residue in the backbone [36], as six Lys in the structure of these peptides make even (М+5Н)5+ ion proton-deficient. The absence of a mobile proton activates alternative mechanism with participation of glutamic acid. Thorough study of the spectra of all peptides (1–11) did not reveal any peaks due to the loss of CO from their protonated molecules with formation of аn ions, although Liu et al. [36] proposed to use this ion as a marker of fragmentation of ranid peptides with the opening of “Rana box.” The data in Table 1 clearly demonstrate that cleavages of amide bonds inside “Rana box” of intact natural peptides in conditions of low-energy CID depend on the sequence of these peptides and the degree of protonation of the precursor ions. For the triggering of informative fragmentation inside “Rana box,” the minimal degree of protonation of the precursor ion is preferable (i.e., conditions of deficit of protons should be realized). The probability of this process becomes higher if basic residues are present at the Cterminus. Alternatively, an acidic residue (Asp or Glu) in the vicinity of “Rana box” may facilitate this process as well.

Conclusion Routine LC-ESI-MS/MS low-energy CID analysis of natural frogs’ skin peptides with disulfide cycle revealed new fragmentation pathway with the opening of S–S loop via amide bond cleavage. This fragmentation becomes quite pronounced in case of proton-deficient precursor ions and in the presence of basic residues at C-terminus. It may also be

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supported by the acidic protons of Asp and Glu residues neighboring “Rana box.” Thus, amino acid sequence inside C-terminal S–S loop may be revealed without actual breaking of the S–S bond, which remains intact while peptide bonds are cleaved. The obtained results demonstrate that the fragmentation pattern of long, natural peptides appears to be much more complicated and less predictable when using theories and schemes, proposed on the basis of studies on short model and tryptic peptides. The possibility of direct CID fragmentation of peptides with disulfide cycle at C-terminus should be taken into account both for manual and automatic de novo sequencing. This feature may considerably increase sequencing efficiency without preliminary disruption of S–S bonds as an additional stage.

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