Electrospray ionization tandem mass spectrometry ...

T.Yu. Samgina et al., Eur. J. Mass Spectrom. 13, 155–163 (2007)

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Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges

T.Yu. Samgina, K.A. Artemenko, V.A. Gorshkov�� and�� A.T. Lebedev ��* Moscow State University, 119992�� Moscow, �� Russia,�� E-mail: [email protected]

M.L. Nielsen, M.M. Savitski�� and�� R.A. Zubarev Uppsala University, Uppsala, Sweden

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges (“Rana box”), reduction of the S–S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS3) experiment was also used to elucidate the sequence inside the “Rana box” after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids. Keywords: FT-ICR-MS, carboxymethylation, disulphide bridge, ESI, peptides, sequencing, Rana ridibunda

Introduction The�� skin secretion �� produced�� by�� granular�� skin�� glands� �� of�� anurans�� in response to a variety of stimuli contains�� a pharmacologically�� -�� active�� peptides�� as �� well as amines and alkaloids�.1–4 Several earlier publications have dealt with the relationship between the structure and bioactivity of host-defense peptides of amphibians. They have been shown to inhibit the growth of a representative of raw pathogenic bacteria, protozoa and fungi.1,5,6 Several peptides of this type revealed anticancer activity, which was not accompanied by hemolytic activity.7,8 Nowadays, over 400 antimicrobial peptides have been isolated from skin secretions of genus Rana frogs.6 Usually,

DOI: 10.1255/ejms.867

they are cationic peptides with a C-terminal CO2H group. A structural peculiarity of the majority of these peptides is a disulfide bridge at the C-terminus,6 resulting in a cycle with seven amino acids (more rarely six, eight or nine members). It is called the “Rana box”.9 The influence of this cycle on the biological activity is not clear so far.6 For some of these peptide reductions, the S–S bond leads to distortion of the three-dimensional configuration accompanied by the loss of antimicrobial activity,10,11 for others, the same procedure does not change their activity.12,13 An important characteristic of peptides–antibiotics of the Rana family involves their simultaneous high activity against both Gram-positive and Gram-negative organisms. A wide spectrum of bioactivities provokes a stable scientific interest towards these compounds as potential pharmaceuticals.1,14,15

ISSN 1469-0667

© IM Publications 2007

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Tandem mass spectrometry (MS/MS) sequencing as well as Edman sequencing of the peptides belonging to genus Rana pose a difficult problem due to the presence of a disulfide bridge at the C-terminus and a rather high molecular mass of peptides (over 2000 Da). A notable number of these peptides has recently been structurally characterized using cDNA cloning techniques.1,16–19 For smaller peptides (less than 1500 Da), despite the presence of the Rana box, Edman degradation produces less reliable results.20 However, larger peptide lengths require preliminary enzymatic digest followed by chromatographic separation of the shorter fragments. Direct sequence identification inside the Rana box has been achieved using electrospray ionization (ESI) MS/MS in the negative ion mode for some bioactive peptides isolated from the Crinia genus (Australia) and for brevinin-1E (R. ridibunda, Russia).21 A combination of the positive and negative ion data for the latter 24-membered antimicrobial peptide gave a complete sequence with the exception of the relative order of the first two residues. Unfortunately, not all peptides give reasonable negative ion mass spectra. In addition, interpretation may be rather difficult due to the ambiguous rationalization of fragmentation in the negative mode. Reduction of the S–S bonds followed by carboxy methylation of cysteine residues with iodoacetamide (IAM) is a useful procedure preceding MS/MS analysis of protein chains connected by S–S bonds.22–24 In addition to IAM, phenylmaleinimide is also used as an alkylation agent.25 In the present study, we report on the sequencing of frog peptides isolated from the skin secretion of Rana ridibunda26 and Rana arvalis27 (Moscow region). The sequences of two� of�� the peptides have not�� been reported�� earlier�. �� Experimental All the solvents were purchased from Panreac (Spain). Trifluoroacetic acid, as well as dithiothreitol and ammonium hydrogen carbonate, were purchased from Acrus (Russia). All chemicals were used without additional purification. Preparation of skin secretions

Male and female Rana Ridibunda and Rana arvalis species were caught in the Moscow region. Adult animals were maintained in captivity at the Biology Department of the Moscow State University under conditions close to natural ones. Secretions were collected from skin glands using mild electrical stimulation28 for 40 s �� using a laboratory electrostimulator, ESL-1, equipped with platinum electrodes. �� The duration of each impulse was 3 ms with an amplitude of 10 V at 50 Hz. The skin secretions were washed from the animals with distilled water while an equal volume of methanol was immediately added to the aqueous solution. The mixtures were centrifuged and filtered through a polymyer thick film (PTF) membrane filter (0.45 μm). The solutions obtained were concentrated using rotary evaporator to a volume of 1 mL and lyophilized.

Sequencing from Novel Skin Peptides from Ranid Frogs

High-performance liquid chromatography (HPLC) separation of crude secretion

1 mg of lyophilized crude secretion was dissolved in 1 mL of distilled water. Peptide separation was performed with an HPLC system (Thermo) by injecting 250 μL of the crude extract into a semi preparative ACCUBOND C �18 (5 µ, 300 A, 4�� × �� 250 mm�� ) reverse-phase chromatographic column equilibrated �� with 10% acetonitrile/aqueous solution containing 0.1% of trifluoroacetic acid�� . Peptides were purified using a linear gradient from 10 to 70% acetonitrile containing 0.1% trifluoroacetic acid. The procedure took 40 min �� at a flow rate of�� 1 mL�� min–1. RP-HPLC experiments were monitored at 214 nm of UV absorption. All fractions eluted after 25 min were collected manually and subsequently lyophilized. The additional HPLC separation was used to obtain individual peptides. The fractions collected, as pointed out above, were separated with the same HPLC system using a 60 min linear gradient from 40 to 65% acetonitrile containing 0.1% trifluoroacetic acid. Disulfide bond reduction and alkylation

HPLC purified and lyophilized active peptide fractions were redispersed in ammonium buffer (100 mM NH4HCO3, pH 8.0) with 10 mM iodoacetamide incubated in the dark for 1 h at 37°C. Prior to treatment with iodoacetamide, dithiothreitol was added at a concentration 4 mM while nitrogen was flushed to provide an inert atmosphere throughout the reaction.29 Amino acids sequencing

All experiments were performed on a 7 tesla hybrid LTQ FT mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) modified with a nano-ESI ion source (Proxeon Biosystems, Odense, Denmark). Highperformance liquid chromatography used on-line with the mass spectrometer consisted of a solvent degasser, nanoflow pump and thermostated microautosampler (Agilent 1100 nanoflow system). A 15 cm fused silica emitter (75 µm inner diameter, 375 µm outer diameter; Proxeon Biosystems) was used as an analytical column. The emitter was packed in-house with a methanol slurry of reverse phase, fully end-capped Reprosil-Pur C18-AQ 3 µm resin (Dr Maisch GmbH, Ammerbuch-Entringen, Germany) using a pressurized “packing bomb” operated at 50–60 bars (Proxeon Biosystems). The samples were dissolved in a cetonitrile / water mixture (1 : 1) with the addition of 0.1% of formic acid and injected into the reversed-phase nano column. Analysis was performed using unattended data-dependent acquisition mode in which the mass spectrometer automatically switches between a low resolution survey mass spectrum, high resolution “zoom” spectrum and consecutive ECD and CID fragmentation of the most abundant detected peptides eluting at that moment from the nano-LC column. A resolving power of 100,000 was used in the zoom MS mode and 50,000 in the MS/MS mode.


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Figure 1. Product ion mass spectrum of the protonated peptide with MW 3517.

Edman degradation

Automated Edman sequencing was performed using a standard procedure30 to confirm the sequences established by means of mass spectrometry. Results and discussion Peptide with the molecular mass of 3516.92 Da

The molecular mass of one of the R. ridibunda peptides measured with the FT-ICR instrument was found to be 3516.92 Da. This molecular mass coincides within ±0.01 Da with that of an antimicrobial peptide identified earlier in R.

esculenta by Edman degradation and called brevinin-2Ec.16. Supposing that the R. ridibunda peptide is brevinin-2Ec, we decided to confirm this hypothesis exclusively by means of mass spectrometry. Figure 1 shows MS/MS spectra of the peptide with a molecular mass of 3517 Da. FT-ICR analysis (Figure 1) demonstrates both y- and b-series in equal proportions. However, the sequence information from inside the “Rana box” remains inaccessible. Due to high mass accuracy, positions of lysine and glutamine were identified as Lys6,8,12,16 and Gln20. On the basis of this information, Mascot identified this peptide as brevinin-2Ec with a very high score of 103.

Figure 2. FT-ICR product ion mass spectrum of peptide with MW 3517 after reduction of the S–S bond with DTT and alkylation with IAM.

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In any case, it was challenging to get the sequence inside the Rana box using MS/MS. Figure 2 represents FT-ICR MS/MS spectrum (positive mode) of this peptide after reduction of the S–S bond with �� dithiothreitol�� and alkylation of the C�� ys residues with iodoacetamide. The molecular mass of the obtained derivative is 3632.98 Da, consistent with the formation of new chemical bonds between the two�� sulphur�� atoms of the former cystine bridge and two acetamide groups. The presence of new electron withdrawing groups attached to cysteines does not seriously modify the fragmentation pattern of the peptide. The most important new aspect involves the extension of the b-series now practically reaching the C-terminus. Ions

b28–b33 demonstrate the following amino acids sequence between C�� ys28 and �� C�� ys34: C�� –K–L–S–G–Q–C–OH. The latter sequence perfectly matches the structure of brevinin-2�� Ec��. The mass difference between ions b32 and b33 corresponds to a glutamine residue. Thus, the peptide of interest with the molecular mass 3516.92 Da isolated from the skin secretion of Rana ridibinda is indeed brevinin-2�� Ec�� reported earlier for R. esculenta.16 This was possible to elucidate with mass spectrometry alone. Peptide with the molecular mass of 2934.55 Da

Figures 3(a) and (b) represent MS/MS spectra of another R. ridibunda peptide before and after S–S bond reduction

(a)

(b)

Figure 3. (a) Product ion mass spectrum of the peptide with MW 2935 (X = I/L) and (b) product ion mass spectrum of the peptide with MW 2935 after S–S bond reduction with DTT and further alkylation with IAM (X = I/L).


with dithiothreitol and further alkylation with iodoacetamide. The peptide of this mass was not reported earlier. The spectrum of the authentic peptide [Figure 3(a)] demonstrates y- and b-series, with the y-series being more pronounced. The absence of fragmentation from the C-terminus indicates possible presence of a disulfide bridge. The lowest mass ion of the y-series (y7, 723.34 Da) corresponds to the entire cystine ring. Compilation of the data yields the amino acid sequence from the N-terminus up to Leu20. Two questions remain open: (i) the composition of the cystine ring and (ii) the order of the two first N-terminal amino acids.

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Reduction of the S–S bond followed by alkylation with iodoacetamide notably increased the number of b-ions (20 versus 12 with the intact S–S bond). Thus, the b-series practically reached the C-terminus and gave the following sequence inside the cycle: C–K–L/I–T–G–T–C–OH. The peptide has a hydroxyl form. This finding is supported by the loss of 178.05 mass units (alkylated cysteine amino acid) from the MH ��+ ion (3051.61 Da) accompanying the cleavage of the peptide bond between �� T�� hr27 and �� C�� ys28 with the formation of b27 ion. Another confirmation involves the loss of a neutral with the mass of 18.01 Da corresponding

Figure 4. (a) Product ion mass spectrum of intact peptide with MW 1874 (X = I/L) and (b) product ion mass spectrum of the peptide with MW 1874 after derivatization (X = I/L).

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to the elimination of a water molecule from MH ��+ [ion m/z 3033.60, Figure 3(b)]. The third residue from the N-terminus was identified as oxidized methionine rather than Phe since its mass in both mass spectra was measured as 147.04 Da and not 147.07 Da. The calculated mass of the oxidized methionine is 147.03 Da. The final confirmation deals with the presence of a peak due to the loss of a methanesulfenic acid molecule (CH3SOH, –64 Da) from the fragment ion y26, containing oxidized methionine (Mo) residue [Figure 3(a)].31–33 Methionine oxidation may take place in solution during sample preparation.31,32 In the present case, the process may proceed during isolation of the secretion and during the LCMS analysis. The authentic peptide with a mass of 15.99 Da lower was also detected. Unfortunately, MS/MS spectra of this peptide are of low quality because of the very low concentration in the initial crude secretion. Anyway, the sequence of five N-terminal residues coincides with that of the peptide with the mass of 2934.55 Da. Due to high resolution capabilities, the positions of lysine and glutamine were identified as Lys7,19,23 and Gln8,21. Thorough inspection of the CAD and ECD spectra of both intact and alkylated peptides reveals the presence of ions w24, w9, w12, d18 and d25 (see example in Figure 6). These ions allow one to distinguish between leucine and isoleucine residues.34,35 The residues in the peptide were identified as Ile6, Leu18, Ile21 and Leu25. Summarizing all data, the following sequence of peptides with the molecular mass of 2934.55 Da was proposed (except for I/L differentiation): (G I/L)MoSTIKGAATNAAVT I/L L NKIQCKLTGTC–OH


Since the level of this peptide in the skin secretion is very low, while it leaves the chromatographic column together with major components, it was impossible to isolate it in quantities reasonable enough to achieve Edman degradation. Peptide with the molecular mass of 1874.10 Da

Figures 4(a) and (b) represent MS/MS spectra of a previously unknown peptide detected in the skin secretion of R. arvalis. The spectrum contains ten y-ions and five b-ions. The intensity of the y-series peaks are higher due to the presence of two lysine residues at the C-terminus. The absence of the fragment ions from y1 to �y6 and from b11 to b17 proves the presence of a disulfide cycle at the C-terminus. Thus, it is possible to establish the sequence of the peptide up to the cystine ring [Scheme at the bottom of Figure 4(a)]. Figure 4(b) represents the CID spectrum of the same peptide after reduction of the S–S bond and cysteine derivatization with IAM. Despite of the lower intensity of the peaks of b and y ions new peaks appeared. These peaks are due to the cleavages inside the former cystine ring (b11, b12, b14, and y4, y5, y6). The peak of ion b16 dominates in the b-series, containing two basic Lys residues at its C-terminus. Also, fragment C�� ys*–�� OH �� is a good leaving group. This fact also contributes to the formation of the b16 ion. Thus carboxymethylation of the initial peptide allowed its complete mass spectrometric sequencing with the exception of two residues (positions 15 and 16). For more reliable identification, a MS3 CID spectrum was recorded for product ion y13 (Figure 5). This experiment confirmed the amino acid sequence inside the former cystine ring, demonstrating the complementary peaks of both

Figure 5. MS3 spectrum of the y13 ion of the peptide with MW 1874 (X = I/L).


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Figure 6. Example of 43 Da losses in the ECD mass spectrum of a peptide with the molecular mass of 1874.10 Da after carboxy methylation. Positions of w13 and w14 ions are shown.

the b- and y-series. The aggregate of all the data (spectra in Figures 4 and 5) gives a complete sequence of the peptide with MW 1874. Due to the high mass accuracy of FT-ICR, the positions of lysine and glutamine were identified as Lys8,15,16. Amino acid in positions 4 and 5 were identified as Leu on the basis of characteristic loss of isopropyl radicals (43.05 Da) from z13 and z14 in the ECD experiment36–38 (Figure 6). The nature of the amino acid in position 9 (Leu vs Ile) was established by Edman degradation to be Leu. Conclusions The efficiency of mass spectrometry of positive ions in the de novo sequencing of peptides with a C-terminal cystine bridge was demonstrated on an example of three peptides isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. Alkylation of the Cys residues with iodoacetamide after the reduction of the –S– S– bond appeared to be very useful for the elucidation of amino acid sequences inside the cystine cycle. Accurate mass measurements enable reliable selection between the particles with the same integral mass, while the ECD technique, in many cases, helps to distinguish between the isomeric leucine and isoleucine residues. References 1.

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Received: 11 April 2007 Accepted: 28 April 2007 Publication: 13 June 2007