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A General Approach to Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry. Anastasia Albert, J. Johannes Eksteen, Johan Isaksson, Myagmarsuren Sengee, Terkel Hansen, and Terje Vasskog Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02115 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Analytical Chemistry

A General Approach to Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry. Anastasia Albert†, J. Johannes Eksteen†, Johan Isaksson‡, Myagmarsuren Sengee†, Terkel Hansen§, Terje Vasskog†*. † Norut Northern Research Institute, 9294 Tromsø, Norway ‡ Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, 9037 Tromsø, Norway § Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, 9037 Tromsø, Norway ABSTRACT: Within the field of bioprospecting, disulfide-rich peptides are a promising group of compounds that has the potential to produce important leads for new pharmaceuticals. The disulfide bridges stabilize the tertiary structure of the peptides and often make them superior drug candidates to linear peptides. However, determination of disulfide connectivity in peptides with many disulfide-bridges has proven to be laborious and general methods are lacking. This study presents a general approach for structure elucidation of disulfide-rich peptides. The method features sequential reduction and alkylation of a peptide on solid phase combined with sequencing of the fully alkylated peptide by tandem mass spectrometry. Subsequently, the disulfide connectivity is assigned based on the determined alkylation pattern. The presented method is especially suitable for peptides that are prone to disulfide scrambling or are unstable in solution with partly reduced bridges. Additionally, the use of small amounts of peptide in lowest nmol range makes the method ideal for structure elucidation of unknown peptides from the bioprospecting process. This study successfully demonstrates the new method for seven different peptides with two to four disulfide-bridges. Two peptides with previous contradicting publications, µ-Conotoxin KIIA and Hepcidin-25, are included and their disulfide connectivity is confirmed in accordance with the latest published results.

INTRODUCTION. The pharmaceutical industry is in constant need of new drug candidates. Especially within the field of antimicrobial compounds the situation is becoming critical worldwide as many bacteria are developing multiresistance to the current treatment. Bioactive peptides found in both animals and plants can be important leads for new pharmaceuticals and are therefore an important group of compounds within the field of bioprospecting. Frequently, bioactive peptides contain disulfide bridges that increase their stability and ensure a well-defined tertiary structure often making them superior to linear peptides as drug candidates.1,2 The bioactivity of cysteine-rich peptides is highly dependent on their tertiary structure3,4 and changes in the disulfide bridge pattern can have a dramatic effect on their activity and stability5,6. Furthermore, exploration of different isomers of the same peptide can lead to more active or more stable peptides with reduced side effects. There is a need for easy and reliable methods for disulfide connectivity assignment of naturally occurring peptides as well as of synthetic analogs. Previous research has demonstrated that it is often difficult to determine the disulfide connectivity leading to laborious experiments for new bioactive peptides.7,8 An additional limitation for any method is the low amount of new peptides isolated in the bioprospecting process.

Numerous methods have been presented in publications ranging from NMR analysis to chemical methods with mass spectrometric analysis. Different NMR techniques, including 1DNMR and 2D-NMR, were successfully used to solve the structure of several peptides.9–11 However, disulfide connectivity cannot be directly assigned using NMR data. Instead, arrangement of possible disulfide bridges has to be derived from the three-dimensional NMR structure. Such indirect methods can lead to difficulties in disulfide bridge assignment for peptides with structural inhomogeneity in solution.5,10,12 For instance, data derived from NMR experiments lead to false assignment of disulfide connectivity in µ-Conotoxin KIIA.13 An alternative structure for the µ-Conotoxin was proposed after evaluation of NMR data in combination with the available crystal structure.11 Based on NMR data, two studies also suggested false disulfide bridge connectivity for the fourbridged human hepcidin14 and its analogue bass hepcidin15. The correct structure was solved by a more complex method of partial reduction in solution followed by proteolytic digestion combined with Edman degradation and confirmed by low temperature NMR.10 Partial reduction in solution for peptides was first introduced by Gray et al.16 and the method was used in numerous studies in modified forms7,17,18. The first publication by Gray et al. demonstrated the procedure for two- and three-bridged pep1

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tides but also identified some limitations of the method. Even when reduction was carried out at low pH, disulfide scrambling still occurred for some peptides and remains an ongoing problem leading to contradicting results for disulfide bridging.19,20 In addition, the study of Gray et al. demonstrated that not all peptides are accessible to partial reduction in solution due to instability of partly reduced forms as was the case for ω-Conotoxin. Echterbille et al. oxidized disulfide bridges in model peptides to avoid scrambling of disulfide bonds during the reduction process.21 The partly oxidized species were separated by ion mobility and analyzed by tandem mass spectrometry. The method was successfully demonstrated for two-bridged peptides but larger peptides with more disulfide bridges could cause separation problems and lead to complex tandem mass spectra of partly oxidized species. Other research groups combined partial reduction in solution with proteolytic digestion10,19 or cleavage after cyanylation22,23 to obtain peptide fragments with preferably one disulfide bridge in each fragment. Both proteolytic digestion and cleavage after cyanylation are carried out at relatively high pH, which can lead to disulfide scrambling.19,23 Alternatively, proteolytic digestion can be carried out with different enzymes that are sufficiently active at low pH. However, problems with insufficient digestion arise when peptides are highly knotted or have closely spaced cysteines. In addition, the described methods often have a complex procedure where purification of the peptide is needed between different techniques. Alternative approaches used direct mass spectrometric fragmentation by collision induced dissociation (CID)8,24 or electron transfer dissociation (ETD)25,26 of proteolytic peptides. Peptides were digested without reduction and peptide fragments with intact disulfide bridges were fragmented to identify the disulfide connectivity. A direct online approach was demonstrated by Cramer et al. with electrochemistry coupled to mass spectrometry.27 Peptides and chromatographed protein digests were partially reduced online in an electrochemical cell and by tandem mass spectrometry. Direct fragmentation of peptides with intact disulfide bridges by CID usually provides complex MS/MS spectra and assignment of connectivity from the spectra becomes challenging.8,24 In addition, the difficulties described above arising from the digestion procedure still remain in both approaches. Although, online approaches are rapid methods and require small sample amounts it is difficult to implement more than one reduction step in the procedure leading to analysis of peptides with partly intact disulfide bridges. In this study, a novel general approach for structure elucidation of disulfide-rich peptides is presented. The method features sequential alkylation combined with sequencing by liquid chromatography tandem mass spectrometry. Reduction and alkylation of disulfide bridges are performed on solid phase with different maleimides and the fully alkylated peptide is submitted to sequencing. The resulting alkylation pattern provides information about the disulfide connectivity in the peptide. The presented procedure is easy to follow, minimizes disulfide scrambling, and produces mass spectra that are relatively easy to interpret. The method is demonstrated for seven different peptides with up to four disulfide bridges including µ-Conotoxin KIIA, ω-Conotoxin GVIA, and Hepcidin-25 that all caused difficulties in previous publications.

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EXPERIMENTAL SECTION. Materials and Reagents. Peptides α-Conotoxin IMI, Tertiapin, and µ-Conotoxin KIIIA were synthesized in-house. Peptides Enterotoxin Stp, ω-Conotoxin GVIA, Defensin NHP1, Hepcidin-25, and native forms of α-Conotoxin IMI and Tertiapin were purchased from Bachem (Budendorf, Switzerland). Tris(2-carboxyethyl)phosphine, N-methylmaleimide, Nethylmalemide, N-propylmaleimide, N-cyclohexylmaleimide, Biotin-maleimide, and thiosalicylic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were purchased from either Sigma-Aldrich or VWR International (Radnor, PA, USA). Ultrapure water was obtained using a RiOs 100 Milli-Q purification system from Merck Millipore (Billerica, MA, USA). Reduction and alkylation were performed on Empore C18 solid phase extraction cartridges (3M, St. Paul, MN, USA). PierceTM Polyacrylamide Desalting Columns (1.8K MWCO, 5 mL) for desalting of peptides were supplied by ThermoFischer Scientific (Waltham, MA, USA). Evaporation of organic solvents was performed with a block heater and a sample concentrator from Stuart (Bibby Scientific, Stone, UK). Peptide synthesis and folding. Peptides α-Conotoxin IMI, Tertiapin, and µ-Conotoxin KIIIA were synthesized on solid phase using a Prelude instrument (Protein Technologies Inc., Tucson, AZ, USA) following standard Fmoc-protocols. Detailed information on synthesis and oxidative folding are provided in SI (cf. section “peptide synthesis and folding” and table S-1). Sample preparation. Purchased or synthesized peptides were stored at -18 ºC. Stock solutions of peptides were prepared in 0.1% formic acid (FA) with a concentration of 0.5 mM. Stock solutions of N-methylmaleimide (NMM), N-ethylmaleimide (NEM), and N-propylmaleimide (NPM) were prepared in acetonitrile (ACN) with a concentration of 2 M and further diluted with ammonium formate buffer (buffer 1, 50 mM, pH 3) for alkylation. N-cyclohexylmaleimide (NCM) was prepared with a concentration of 100 mM in ACN and Biotinmaleimide (BM) with a concentration of 20 mM in water/acetic acid (50:50, v/v). The reduction solution with Tris(2carboxyethyl)phosphine (TCEP) was prepared freshly on a daily basis in buffer 1 and further diluted for the reduction procedure. A stock solution of thiosalicylic acid (TA) was prepared in purified water with a concentration of 0.5 M. All stock solutions were stored at 4 ºC when not in use. Method development for reduction conditions. Reduction conditions were optimized for each peptide during method development. Three aliquots of the peptide of interest, each containing 0.5 nmol of peptide, were submitted to a 1-minreduction on an equilibrated solid phase extraction (SPE) cartridge with the three different TCEP concentrations 2, 10, and 50 mM. The partly reduced peptide was alkylated with the maleimide NMM and eluted from solid phase for LC-MS analysis. Reduction ratios were calculated based on peak area in the extracted ion chromatograms of partly reduced peptide species. For the first step in the main procedure, the TCEP concentration and reduction time providing highest reduction ratio for one disulfide bridge were selected. For the second and third step on solid phase, TCEP concentration and reduction time leading to highest reduction ratios for two and three 2


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bridges, respectively were chosen. In addition, the TCEP concentration was scaled up according to the amount of peptide used in the main procedure. Sequential reduction and alkylation on solid phase. In the main procedure, reduction and alkylation of a peptide were accomplished sequentially with different maleimides on reversed phase C18 solid phase. The method consisted of several reduction and alkylation steps according to the number of disulfide bridges in a peptide. For the first step, an aliquot of a peptide stock solution was diluted with buffer 1 to 500 µL and loaded on an equilibrated SPE cartridge. For the reduction, 200 µL of TCEP of varying concentrations were applied and let to soak for varying times according to method development for each peptide. After the reduction, most of the TCEP was washed off the solid phase with a mixture of buffer 1 and ACN followed by buffer 1. The ratio of buffer 1 and ACN for washing was 15% below the concentration of ACN at which the peptide eluted from the SPE column. For alkylation, a volume of 200 µL of the first maleimide NMM (20 mM in buffer 1) was applied onto the cartridge immediately after reduction and washing and let to soak for one hour. After the first reduction and alkylation step, the maleimide was removed depending on peptide properties. Peptides were classified in three groups as follows: hydrophobic peptides, large peptides and small hydrophilic peptides. For hydrophobic peptides, which eluted from the SPE cartridge with more than 30% ACN, NMM and the reaction product TCEP-NMM were removed from the SPE cartridge with 2x 500 µL buffer 1/ACN (85:15, v/v). The remaining peptide was washed with 500 µL buffer 1. The following reduction and alkylation steps were performed on the same SPE cartridge. Larger peptides with a molecular weight higher than 2 kDa were eluted from the SPE cartridge with 300 µL buffer 1/ACN (20:80, v/v) and desalted on a PierceTM Polyacrylamide Desalting column. The desalted peptide was loaded onto a freshly equilibrated SPE cartridge for the second step. In the third strategy for small hydrophilic peptides, the peptide was eluted from the SPE cartridge with 300 µL buffer 1/ACN (20:80, v/v) and the ACN evaporated in the sample concentrator. To remove the remaining NMM, TA (10 µL) was added and the mixture incubated at room temperature for 30 min. After the incubation, the mixture was diluted to 500 µL and loaded onto a freshly equilibrated SPE cartridge. Excess TA and the reaction product TA-NMM were washed off with a mixture of buffer 1 and ACN followed by buffer 1. Subsequently, the cleaned partly reduced and alkylated peptide was subjected to additional reduction and alkylation steps on solid phase according to the number of disulfide bridges. In the second and third step, NEM and NPM, respectively, were used for alkylation and removed according to one of the procedures described above. For the final reduction/alkylation step, the peptide was eluted from the SPE cartridge with 300 µL buffer 1/ACN (20:80, v/v) and incubated with 5-10 µL TCEP (100 mM) for one hour followed by incubation with 6-12 µL NCM solution or 40-80 µL BM solution for one to several hours. The organic solvent was removed from the mixture in the sample concentrator. The completely alkylated peptide was submitted to LCMS and LC-MS/MS analysis. LC-MS and LC-MS/MS analysis. Reduced and alkylated peptides were analyzed either on an ACQUITY CSH C18 UPLC column (Column 1, 2.1 mm x 150 mm, 1.7 µm) or on

an ACQUITY BEH C18 UPLC column (Column 2, 1.0mm x 150 mm, 1.7 µm) installed in an ACQUITY UPLC I-Class system (Waters, Milford, MA, USA) and coupled to a Xevo G2 Q-TOF mass spectrometer (Waters). Varying binary gradients were run with solvent A (purified water + 0.1% FA) and solvent B (ACN + 0.1% FA) at flow rates 600 µL/min and 200 µL/min for column 1 and 2, respectively. The column temperature was set to 65 ºC for both columns. For LCMS/MS analysis, single ion traces of different peptide charge states were selected and fragmented at different collision energies according to a manually optimized diagram for each charge state and ion mass (data not shown). Gradients and parameters for LC-MS and LC-MS/MS analyses are provided in supporting information (SI, cf. table S-2 and table S-3). Best fragment ion coverage in MS/MS analyses was obtained with a collision energy ramp at adjusted cone voltage for each peptide charge state. Parameters for cone voltage and collision energy ramps for each alkylated peptide are included in SI (cf. table S-4). Data processing. Raw tandem mass spectra were mean centered and averaged across the peak width for each alkylation pattern. Theoretical fragment masses for reduced peptides were generated with Protein Prospector v 5.14.4 (University of California, San Francisco, USA) and Microsoft Excel 2010 (Microsoft, Redmond, USA) was used to generate fragment masses for alkylated peptides with different maleimides. Alkylation patterns were identified from characteristic combinations of y-type, b-type, and a-type ions. Fragments in mass spectra were identified with mass accuracy below 20 ppm, typically below 10 ppm for masses below 1000 Da, and a minimum signal-to-noise ratio of three. Grand average of hydropathicity (GRAVY) was calculated with ExPASy ProtParam (SIB Swiss Institute of Bioinformatics). NMR analysis and structure calculation. Isomers I, II, and III of Tertiapin were analyzed with NMR. All experiments were conducted on a 600 MHz Varian Inova spectrometer (Agilent, St. Clara, CA, USA) equipped with an inverse HCN probe with cryogenic enhancement for 1H. Approximately 0.4 – 0.8 µmol of peptide were dissolved in 110 µL ultra-pure water and 10 µl D2O and transferred to a 3 mm Shigemi-tube matched for D2O. All spectra were acquired with presaturation on the water and four purge cycles, except for the 1H,15N-HSQC which was acquired with 3919 watergate. All spectral parameters are summarized in SI (cf. table S-5). The spectral assignments for Tertiapin-I and –II are summarized in SI (cf. tables S-6 and S-7). The fold was determined by molecular modelling using Xplor-NIH 2.35, running a standard simulated annealing protocol (anneal.inp) between 3500 and 100 K. Detailed information on implemented constraints is included in SI (cf. table S-8).

RESULTS AND DISCUSSION. Disulfide-rich peptides. In this study, structure elucidation of disulfide bridges is demonstrated for seven peptides with two to four disulfide bridges including different isomers for two of the peptides. Detailed information on the peptides size, hydropathicity, and number of disulfide bridges is given in table 1. Table 1.

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Additional information in form of a chromatogram of all peptides is included in SI (cf. figure S-1). Three of the peptides, αConotoxin, Tertiapin, and µ-Conotoxin, were synthesized inhouse and were included with two isomers of α-Conotoxin, three isomers of Tertiapin, and one isomer of µ-Conotoxin. Principle and workflow. The principle for sequential reduction and alkylation of a peptide on solid phase is shown in figure 1 for a representative peptide with two disulfide bridges. Figure 1.

Adsorption on solid phase immobilizes the peptide and presumably restricts the accessibility of TCEP to some of the disulfide bridges in the peptide. Depending on the peptides orientation on the solid phase, some disulfide bridges will be more exposed to TCEP than others and therefore will be reduced faster and alkylated with one maleimide. Subsequent application of TCEP reduces the remaining disulfide bridge, which is alkylated with a different maleimide. Ideally, only one bridge is reduced and alkylated in each step. During the procedure, disulfide scrambling is minimized because of the restrained orientation of the peptide. Thus, the resulting fully alkylated peptide is labeled with different maleimides according to its given disulfide connectivity. In the final reaction mixture, the two-bridged peptide can be present with two homologous alkylation patterns resulting from the same disulfide bridge connectivity. For peptides with more disulfide bridges more alkylation patterns are possible. In which order the disulfide bridges are reduced and how many alkylation patterns are produced depends on the peptide and in which way it can be oriented on the solid phase. The general workflow for structure elucidation can be applied to synthesized and folded peptides with determined amino acid sequence or to peptides obtained from the bioprospecting process with unknown amino acid sequence (cf. figure 2). Figure 2.

In the presented workflow, peptides with unknown sequence are first analyzed by means of LC-MS and de novo sequenced by LC-MS/MS to determine the amino acid sequence and number of disulfide bridges. Accordingly, peptides with known or identified amino acid sequence are subjected to a short method development on solid phase to determine the appropriate TCEP concentration and reduction time. In the following main procedure, sequential reduction and alkylation of the peptide on solid phase is performed. After alkylation of the partly reduced peptide with one maleimide in the first step, excess reagents are removed. Depending on the peptide size and hydropathicity, three different approaches can be used to remove the reagents (cf. experimental section, section “sequential reduction and alkylation on solid phase”). Subsequently, the reduction and alkylation procedure is repeated on solid phase in several steps with different maleimides till all disulfide bridges are reduced and alkylated. In the final step, reduction and alkylation can be carried out in solution since there is no risk of disulfide scrambling. The fully alkylated peptide is submitted to LC-MS/MS sequencing to determine the location of different maleimides and hence the disulfide connectivity. Sequencing of several alkylation patterns of the same peptide provides additional confirmation on its disulfide structure. For six of the analyzed peptides in this study, alkyla-

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tion with two different maleimides and sequencing of different alkylation patterns yielded enough information to solve the disulfide connectivity. However, other peptides may not provide enough information with just two reduction and alkylation steps as was the case for µ-Conotoxin. Therefore, each peptide was reduced and alkylated sequentially with up to four maleimides according to the number of disulfide bridges. Method development. The method development procedure was optimized for minimal workload and required amount of peptide. A representative plot of reduction ratios for the three Ter isomers is displayed in figure 3. Figure 3.

Isomers of Tertiapin with different disulfide connectivity show different reduction ratios, ranging from 29% to 55%, for one disulfide bridge. The final TCEP concentration and reduction times for the main procedure were derived from the graphs obtained in method development. For example, the concentration of the singly reduced species rapidly decreases with higher TCEP concentrations for the isomers Tertiapin-II and Tertiapin-III. Therefore, the reduction time was decreased with the lowest TCEP concentration for the main procedure. In general, decreasing the reduction time by a factor had the same effect on the reduction rate as decreasing the TCEP concentration by the same factor (data not shown). Typically, reduction with TCEP on solid phase yielded approximately 30 – 40% of singly reduced peptide. One exception was the threebridged ω-Conotoxin that proved to be impossible to partly reduce in previous publications.16 In this study, a reduction ratio of 14% for one disulfide bridge was achieved, which was sufficient to solve the connectivity. An overview of the amount of peptide used, TCEP concentration and reduction time for all peptides is shown in table 2. Table 2. The amount of peptide used in the main procedure varied according to the ionization efficiency and number of steps needed for sequential reduction and alkylation of all disulfide bridges. In general, only 2.5-5 nmol of a peptide were sufficient for alkylation with two maleimides and up to 12.5 nmol of a peptide were used for alkylation with up to four maleimides. Case 1: two-bridged peptides. In this study, peptides with two disulfide bridges include Tertiapin and α-Conotoxin. Structure elucidation of two-bridged peptides is comparatively easy. In theory, four cysteines can connect in three different ways resulting in six theoretical alkylation patterns with two different maleimides. Consequently, the fully alkylated peptide with two different maleimides can be alkylated in two homologous alkylation patterns for a given disulfide connectivity. All isomers of Tertiapin covering three possible disulfide bridge connectivities were subjected to sequential reduction and alkylation. A representative chromatogram of fully alkylated Tertiapin-I with the two maleimides NMM and NCM is presented in figure 4. Figure 4.

The peptide was alkylated in two homologous alkylation patterns, which each split in two distinct peaks in the chromato4


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gram. Presumably, the splitting is caused by different conformers and was observed for several peptides throughout the study. With sequencing by LC-MS/MS, the alkylation patterns were assigned based on combination of characteristic fragments. Fragments identified as y-type ions with m/z 613.349, 635.333, and 842.775 and b-type ions with m/z 329.091 and 881.339 extracted from the first peak in the chromatogram in figure 4 identified the alkylation pattern C3-NMM/C5NCM/C14-NMM/C18-NCM for Tertiapin-I. Detailed information on identified fragments and MS/MS spectrum are included in SI (cf. figure S-19 and table S-9). The determined pattern corresponds to the connectivity C3C14/C5-C18, which was also found in the native form of Tertiapin (data not shown). The same disulfide connectivity resulted from the second homologous alkylation pattern in the chromatogram. Identified fragments are included in SI (cf. figure S-20, table S-10). With the same method, the isomers Tertiapin-II and Tertiapin-III were sequentially reduced and alkylated with maleimides NMM and NCM. The peptide Tertiapin-III produced two homologous alkylation patterns. The chromatogram of alkylated Tertiapin-III and identified fragment ions can be found in SI (cf. figures S-2, S-21, and S22, tables S-12 and S-13). Based on the determined alkylation patterns, the disulfide connectivity for Tertiapin-III was assigned to C3-C18/C5-C14. In contrast, Tertiapin-II was alkylated with only one alkylation pattern (SI, cf. figures S-3 and S23, table S-11) leading to the unusual connectivity C3C5/C14-C18. The peptide Tertiapin was used to develop and optimize the procedure for reduction and alkylation on solid phase. To validate obtained structures for its different isomers, an additional independent method was implemented. All synthesized isomers of Tertiapin were available in sufficient amounts for analysis with NMR. From a first qualitative assessment of the 1D proton spectra (SI, cf. figure S-52), it was possible to distinguish Tertiapin-I as being well folded with clearly separated resonances in the 9.0 – 6.0 PPM region. Assignment and a crude structure determination to prove the disulfide bridge connectivity of Tertiapin-I was straightforward and confirmed that this isomer was the native fold with the connectivity C3C14/C5-C18. The structure and spectra were identical to the originally published NMR structure28 (SI, cf. figure S-53A to S-53B). In contrast, Tertiapin-II showed typical unfolded appearance with all amide protons clustered between 7.5 and 8.5 PPM, which corresponds well with the expected behavior of a C3-C5/C14-C18 disulfide pattern. Tertiapin-II was assigned in the same way as Tertiapin-I and structural parameters were extracted. The Tertiapin-II isomer displayed spectra typical for random coil peptides and proteins showing very few long range inter-residual correlations. The C-terminal is the only structured part of the peptide with the only significant long range contact found between the β-protons of C14 and C18 thus confirming the C3-C5/C14-C18 disulfide pattern (SI, cf. figure S-53D). The spectra of Tertiapin-III indicated heterogeneity and the spectra quality was not adequate to determine the disulfide pattern. In summary, NMR data could confirm correct assignment of disulfide bridges by the introduced chemical method in two of the three isomers. By elimination, Tertiapin-III was assigned as the third possible isomer with the C3-C18/C5-C14 disulfide pattern.

Synthesis and oxidative folding of α-Conotoxin yielded only two isomers with different disulfide connectivity. Both isomers, α-Conotoxin-I and α-Conotoxin-II, were sequentially reduced and alkylated with maleimides NMM and NCM. For α-Conotoxin-I, two homologous alkylation patterns determined the disulfide connectivity C2-C8/C3-C12 (SI, cf. figures S-4, S-24, and S-25, tables S-14 and S-15). The same connectivity was found in the native form of α-Conotoxin (data not shown). The non-native α-Conotoxin-II was found to contain the disulfide connectivity C2-C12/C3-C8, which was confirmed with two homologous alkylation patterns (SI, cf. figures S-5, S-26, and S-27, tables S-16 and S-17). Interestingly, only two isomers were produced for α-Conotoxin in oxidative folding whereas Tertiapin yielded isomers with all three possible connectivities. This finding demonstrates that disulfide bridges between neighboring cysteines, as found in αConotoxin, are not favorable but readily form between cysteines separated by one amino acid as present in Tertiapin. Case 2: three-bridged peptides. The number of possible isomers for three-bridged peptides increases fivefold compared to peptides with only two bridges. Consequently, data evaluation becomes more extensive with an increased number of alkylation patterns but at the same time, it provides more information on disulfide connectivity. The three-bridged peptides in this study, µ-Conotoxin, Enterotoxin, ω-Conotoxin, and Defensin, cover a relatively wide range in size and hydropathicity. The number of amino acids in the sequences ranges from 16 in µ-Conotoxin to 30 in Defensin whereby µConotoxin is hydrophilic and the other peptides are relatively hydrophobic with Enterotoxin displaying the highest hydrophobicity (cf. table 1). Nevertheless, all peptides were accessible to structure elucidation by the described method. The in-house synthesized µ-Conotoxin was folded following two different oxidative methods yielding the same major product. The procedures for oxidative folding were taken from two publications (SI, cf. table S-1) stating different disulfide connectivity for µ-Conotoxin.11,13 Products from both folding methods were sequentially reduced and alkylated with the maleimides NMM, NEM, and NCM. Sequencing with LCMS/MS of the singly formed alkylation pattern C1-NMM/C2NEM/C4-NCM/C9-NEM/C15-NMM/C16-NCM (SI, cf. figure S-6) provided the disulfide connectivity C1-C15/C2-C9/C4C16 for both products. The determined disulfide connectivity agrees with the disulfide pattern stated by Poppe et al. for the naturally occurring µ-Conotoxin11. Detailed information on identified fragments for the product folded according to the procedure from Poppe et al. is included in SI (cf. figure S-28 and table S-18). The three-bridged Enterotoxin was sequentially alkylated with the maleimides NMM, NEM, and NCM resulting in three homologous alkylation patterns (SI, cf. figure S-7). Identification of the pattern C5-NEM/C6-NCM/C9-NMM/C10NEM/C14-NCM/C17-NMM with LC-MS/MS sequencing provided enough information to solve the disulfide connectivity as C5-C10/C6-C14/C9-C17 (SI, cf. figure S-29 and table S19). The additional homologous alkylation patterns confirmed this result (SI, cf. figures S-30 and S-31, tables S-20 and S21). Alternatively, it was possible to determine the connectivity of Enterotoxin by sequential alkylation with two maleimides NMM and NCM. In this experiment, different alkylation patterns were produced with two patterns alkylated with 5



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two NMM/ four NCM and two patterns alkylated with four NMM/ two NCM (SI, cf. figures S-8 and S-9). Sequencing of the alkylated peptide yielded the same connectivity for Enterotoxin as described above (SI, cf. figures S32 to S-35, tables S22 to S-25). Three-bridged ω-Conotoxin is a difficult candidate for partial reduction in solution.16 In the present study, disulfide bridges in the peptide did not reduce under mild reduction conditions or reduced completely with higher TCEP concentration in solution (data not shown). However, partial reduction with TCEP and sequential alkylation with NMM, NEM, and NCM was successful when performed on solid phase. The connectivity was identified and confirmed with two homologous alkylation patterns as C1-C16/C8-C19/C15-C-26 (SI, cf. figures S-10, S-36, and S-37, tables S-26 and S-27). The two alkylation patterns for ω-Conotoxin were C1-NMM/C8NCM/C15-NEM/C16-NMM/C19-NCM/C26-NEM and C1NMM/C8-NEM/C15-NCM/C16-NMM/C19-NEM/C26-NCM suggesting that the disulfide bridge between C1 and C16 is reduced first. Presumably, after reduction of this first bridge under harsh conditions the peptide becomes unstable in solution leading to complete reduction. The same result for disulfide connectivity in ω-Conotoxin was obtained with sequential alkylation with the two maleimides NMM and NCM. Detailed information for the resulting three alkylation patterns is included in SI (cf. figures S-11, S-12, S-38, S-39, and S-40, tables S-28 to S-30). For Defensin, two homologous alkylation patterns were identified after sequential alkylation with the maleimides NMM, NEM, and NCM (SI, cf. figure S-13). Additional alkylation patterns for Defensin were formed but coeluted in the chromatogram and were discarded from data evaluation. In this case, two different disulfide bridges were reduced first resulting in two patterns C2-NMM/C4-NCM/C9-NEM/C19-NCM/C29NEM/C30-NMM and C2-NEM/C4-NMM/C9-NCM/C19NMM/C29-NCM/C30-NEM (SI, cf. figures S-41 and S-42, tables S-31 and S-32). Both patterns derive from the disulfide connectivity C2-C30/C4-C19/C9-C29. With two maleimides, NMM and NCM, the same reduction order lead to two alkylation patterns confirming the disulfide connectivity in Defensin (SI, cf. figures S-14, S-43, and S-44, tables S-33 and S-34). Case 3: four-bridged peptides. Peptides with eight cysteines can form four disulfide bridges leading to 105 different possible isomers. Thus, when using four different maleimides for sequential alkylation, a total number of 2520 alkylation patterns are theoretically possible. Sequential alkylation with only two maleimides reduces the number of possible alkylation patterns to 28. Therefore, the four-bridged Hepcidin was first alkylated with two maleimides and the information used to reduce the number of possible alkylation patterns in the data evaluation with four maleimides. In previous publications, contradicting results for the disulfide connectivity in Hepcidin were reported.10,14,15 In this study, Hepcidin was first sequentially reduced and alkylated with two maleimides NMM and BM. The maleimide BM was chosen over NCM to achieve a better separation of the alkylated peptide species during chromatography. The peptide produced different alkylation patterns whereby two patterns were labeled with two NMM/ six BM, two patterns with four NMM/ four BM, and one pattern with six BM/ two NMM (SI, cf. figures S-15 to S-17). All alkylation patterns were se-

Page 6 of 11

quenced with LC-MS/MS and the connectivity of Hepcidin assigned as C7-C23/C10-C13/C11-C19/C14-C22 (SI, cf. figures S-45 to S-49, tables S-35 to S-39). In this case, alkylation with two maleimides provided enough information to determine the complete disulfide structure. Nevertheless, alkylation of Hepcidin was undertaken with four different maleimides NMM, NEM, NPM, and BM to confirm the connectivity in a second experiment. In the data evaluation for alkylation patterns with four maleimides, information from alkylation with two maleimides was used to reduce the number of theoretically possible alkylation patterns. The bridge between C7 and C23 was set as given reducing theoretically possible alkylation patterns to four relatively small data sets where the C7-C23 bridge can be labeled with NMM, NEM, NPM or BM. In the chromatogram of Hepcidin, two alkylation patterns were identified where C7-C23 was alkylated with NPM and BM (SI, cf. figure S-18). The alkylation patterns were C7-BM/C10-NMM/C11-NEM/C13-NMM/C14NPM/C19-NEM/C22-NPM/C23-BM and C7-NPM/C10NMM/C11-NEM/C13-NMM/C14-BM/C19-NEM/C22BM/C23-NPM (SI, cf. figures S-50 and S-51, tables S-40 and S-41). Both patterns derive from the same disulfide connectivity and confirm the results obtained with two maleimides. The determined disulfide structure agrees with the disulfide pattern of Hepcidin published by Jordan et al. for the naturally occurring isomer10. In general, many homologous alkylation patterns of a peptide can cause separation problems in LC leading to coeluting peaks. With four different maleimides, Hepcidin produced more than three homologous alkylation patterns from which two were identified. In future experiments, improved LC resolution is necessary for peptides with many alkylation patterns, which could be achieved by, for example, changing the column dimensions, column chemistry, or both. Alternatively, a combination of LC with ion mobility MS could provide an additional dimension and improve the resolution of homologous alkylation patterns.

CONCLUSIONS. A new general method based on sequential alkylation on solid phase and mass spectrometry was presented for structure elucidation of cysteine-rich peptides. This improved procedure for assignment of disulfide connectivity was successfully demonstrated for seven different peptides with up to four disulfide bridges. Thereby, isomers with different disulfide bridge connectivities were readily distinguished. All elucidated peptides were in agreement with the latest available information on their disulfide structures. Particularly, the peptides µ-Conotoxin, ω-Conotoxin, and Hepcidin, which proved to be difficult in previous publications, were analyzed successfully and confirmed the latest published results for their disulfide connectivity. The presented method is especially suitable for peptides that are prone to disulfide scrambling or are unstable in solution with partly reduced bridges. Adsorption of the peptide on solid phase stabilizes the partly reduced peptide and allows accessing the disulfide bridges one at a time. The solid phase thereby can be easily adapted to the peptide properties. Additionally, this method requires only small amounts of a peptide in the lowest nmol range making it ideal for structure elucidation of unknown disulfide-rich peptides from the bioprospecting process. With automated instrumentation for 6


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solid phase extraction the reduction and alkylation procedure can be optimized for time efficiency. As the number of disulfide bridges increases in a peptide, the number of theoretically possible isomers grows exponentially leading to extensive data evaluation. For peptides with a high number of disulfide bridges software-assisted assignment of alkylation patterns can be implemented.

ASSOCIATED CONTENT

(14) (15)

(16) (17)

(18)

Supporting Information Supporting information provides chromatograms from LCMS/MS analysis for each alkylated peptide, detailed information on peptide synthesis, tables with LC-MS and LC-MS/MS parameters, detailed information on NMR analysis, tables with peptide fragments from LC-MS/MS analysis. (PDF)

(19)

AUTHOR INFORMATION

(22) (23)

Corresponding Author

(20)

(21)

*E-mail: [email protected]

(24)

Author Contributions

(25)

All authors have given approval to the final version of the manuscript.

(26) (27)

Notes The authors declare no competing financial interest.

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Olivera, B. M. Biochemistry 2005, 44, 7259–7265. Hunter, H. N.; Bruce Fulton, D.; Ganz, T.; Vogel, H. J. J. Biol. Chem. 2002, 277, 37597–37603. Lauth, X.; Babon, J. J.; Stannard, J. A.; Singh, S.; Nizet, V.; Carlberg, J. M.; Ostland, V. E.; Pennington, M. W.; Norton, R. S.; Westerman, M. E. J. Biol. Chem. 2005, 280, 9272–9282. Gray, W. R. Protein Sci. 1993, 2, 1732–1748. Nair, S. S.; Nilsson, C. L.; Emmett, M. R.; Schaub, T. M.; Gowd, K. H.; Thakur, S. S.; Krishnan, K. S.; Balaram, P.; Marshall, A. G. Anal. Chem. 2006, 78, 8082–8088. Bingham, J.-P.; Broxton, N. M.; Livett, B. G.; Down, J. G.; Jones, A.; Moczydlowski, E. G. Anal. Biochem. 2005, 338, 48– 61. Yen, T.-Y.; Yan, H.; Macher, B. A. J. mass Spectrom. 2002, 37, 15–30. Harris, P. W. R.; Yang, S.-H.; Molina, A.; López, G.; Middleditch, M.; Brimble, M. A. Chem. - A Eur. J. 2014, 20, 5102–5110. Echterbille, J.; Quinton, L.; Gilles, N.; De Pauw, E. Anal. Chem. 2013, 85, 4405–4413. Wu, J.; Watson, J. T. Protein Sci. 1997, 6, 391–398. Qi, J.; Wu, J.; Somkuti, G. A.; Watson, J. T. Biochemistry 2001, 40, 4531–4538. Gupta, K.; Kumar, M.; Balaram, P. Anal. Chem. 2010, 82, 8313–8319. Clark, D. F.; Go, E. P.; Desaire, H. Anal. Chem. 2013, 85, 1192–1199. Wu, S.-L.; Jiang, H.; Lu, Q.; Dai, S.; Hancock, W. S.; Karger, B. L. Anal. Chem. 2009, 81, 112–122. Cramer, C. N.; Haselmann, K. F.; Olsen, J. V.; Nielsen, P. K. Anal. Chem. 2016, 88, 1585–1592. Xu, X.; Nelson, J. W. Proteins 1993, 17, 124–137.

ACKNOWLEDGMENT We are grateful to Troms County Council for financial support through the project TFK2013-252. The students Merete Kveli Moen and Regasa Tulu Chala are gratefully acknowledged.

REFERENCES (1) (2)

(3) (4) (5)

(6) (7)

(8) (9)

(10)

(11) (12)

(13)

Hancock, R. E. W.; Sahl, H.-G. Nat. Biotechnol. 2006, 24, 1551–1557. Northfield, S. E.; Wang, C. K.; Schroeder, C. I.; Durek, T.; Kan, M.-W.; Swedberg, J. E.; Craik, D. J. Eur. J. Med. Chem. 2014, 77, 248–257. Flinn, J. P.; Pallaghy, P. K.; Lew, M. J.; Murphy, R.; Angus, J. A.; Norton, R. S. Biochim. Biophys. Acta 1999, 1434, 177–190. Hocquellet, A.; Le Senechal, C.; Garbay, B. Peptides 2012, 36, 303–307. Dutton, J. L.; Bansal, P. S.; Hogg, R. C.; Adams, D. J.; Alewood, P. F.; Craik, D. J. J. Biol. Chem. 2002, 277, 48849– 48857. Gehrmann, J.; Alewood, P. F.; Craik, D. J. J. Mol. Biol. 1998, 278, 401–415. Han, Y.-H.; Wang, Q.; Jiang, H.; Liu, L.; Xiao, C.; Yuan, D.-D.; Shao, X.-X.; Dai, Q.-Y.; Cheng, J.-S.; Chi, C.-W. FEBS J. 2006, 273, 4972–4982. Hung, C.-W.; Jung, S.; Grötzinger, J.; Gelhaus, C.; Leippe, M.; Tholey, A. J. Proteomics 2014, 103, 216–226. Fadel, V.; Bettendorff, P.; Herrmann, T.; de Azevedo Jr, W. F.; Oliveira, E. B.; Yamane, T.; Wüthrich, K. Toxicon 2005, 46, 759–767. Jordan, J. B.; Poppe, L.; Haniu, M.; Arvedson, T.; Syed, R.; Li, V.; Kohno, H.; Kim, H.; Schnier, P. D.; Harvey, T. S.; Miranda, L. P.; Cheetham, J.; Sasu, B. J. J. Biol. Chem. 2009, 284, 24155–24167. Poppe, L.; Hui, J. O.; Ligutti, J.; Murray, J. K.; Schnier, P. D. Anal. Chem. 2012, 84, 262–266. Heck, S. D.; Kelbaugh, P. R.; Kelly, M. E.; Thadeio, P. F.; Saccomano, N. A.; Stroh, J. G.; Volkmann, R. A. J. Am. Chem. Soc. 1994, 116, 10426–10436. Bulaj, G.; West, P. J.; Garrett, J. E.; Watkins, M.; Marsh, M.; Zhang, M.-M.; Norton, R. S.; Smith, B. J.; Yoshikami, D.;

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Page 8 of 11

Table 1. Peptide properties including hydropathicity (GRAVY), molecular weight, number of amino acids, and number of disulfide bridges. Peptide

GRAV Y

Mol. weight [Da]

Length [aa]

α-Conotoxin IMI

-0.37

1351

12

GCCSDPRCAWRC-NH2

2

Tertiapin

-0.11

2455

21

ALCNCNRIIIPHMCWKKCGKK-NH2

2

µ-Conotoxin KIIIA

-0.71

1883

16

CCNCSSKWCRDHSRCC-NH2

3

Enterotoxin STp

0.52

1972

18

NTFYCCELCCNPACAGCY

3 3

Amino acid sequence

Disulfide bridges

ω-Conotoxin GVIA

-0.89

3037

27

CKSXGSSCSXTSYNCCRSCNXYTKRCYNH2

Defensin HNP-1

0.3

3442

30

ACYCRIPACIAGERRYGTCIYQGRLWAF CC

3

Hepcidin-25

0.39

2789

25

DTHFPICIFCCGCCHRSKCGMCCKT

4

8


Page 9 of 11

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Figure 3. Method development for TCEP concentration and reduction time on solid phase for A) Tertiapin-I, B) TertiapinII, and C) Tertiapin-III with a reduction time of 1 min.

Figure 1. Principle for sequential reduction and alkylation with two different maleimides on reversed phase C18 solid phase for a two bridged peptide. The Peptide is alkylated with two homologous alkylation patterns.

Figure 2. General workflow for structure elucidation of disulfiderich peptides. The procedure includes peptides with unknown amino acid sequence and synthesized peptides with determined amino acid sequence.

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Table 2. Amount of peptide used, concentration of TCEP and reduction times on solid phase for different peptides. Peptide amount and TCEP concentration marked with * were used when alkylation was performed with two maleimides.

Peptide

Amount [nmol]

α-Conotoxin-I

2.5

α-ConotoxinII

2.5

1. Reduction step on solid phase



c (TCEP) [mM]

Reduction time [min]

c (TCEP) [mM]


c (TCEP) [mM]


25

1

---

---

---

---

1

---

---

---

---

25

Tertiapin-I

2.5

50

1

---

---

---

---

Tertiapin-II

2.5

5

0.5

---

---

---

---

Tertiapin-III

2.5

5

0.5

---

---

---

---

µ-Conotoxin

2.5*/ 5

25*/ 50

1

50

2

---

---

Enterotoxin

5*/ 10

125*/ 250

1

250

2

---

---

ω-Conotoxin

5*/ 12.5

50*/ 125

3

250

3

---

---

Defensin

2.5*/ 5

125*/ 250

1

250

2

---

---

Hepcidin

5*/12.5

10*/ 25

0.5

125

1

100

1

Figure 4: Chromatogram of Tertiapin-I alkylated with NMM and NCM. Two different alkylation patterns were identified for the peptide. Both patterns display split peaks in the chromatogram, which is presumably caused by different conformers.

10


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General Approach To Determine Disulfide ... - ACS Publications

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