Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.
Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of µ-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.
The use of bioactive peptides in pharmaceutical research and development is highly recognized, because they represent potent and highly selective compounds for specific biological targets1. For their bioactivity, however, the three-dimensional structure is of great importance in order to perform structure-activity relationship studies2,3,4. Apart from the primary amino acid sequence that influences the overall conformation, disulfide bonds significantly stabilize the structure of cysteine-rich peptides5. Multiple disulfide-bridged peptides include conotoxins such as µ-PIIIA from Conus purpurascens which contains six cysteines in its sequence. This high cysteine content theoretically allows the formation of 15 disulfide isomers. The correct disulfide connectivity is very important for the biological activity6,7. However, the question that arises is whether there is more than one bioactive conformation of naturally occurring peptides and if so, which of those isomers possesses the highest biological activity? In the case of µ-conotoxins, the biological targets are voltage-gated sodium ion channels, and µ-PIIIA in particular is most potent for subtypes NaV1.2, NaV1.4 and NaV1.73.
The synthesis of disulfide-bridged peptides can be achieved using various methods. The most convenient method for the formation of disulfide bonds within a peptide is the so-called oxidative self-folding approach. Here, the linear precursor of the desired cyclic peptide is synthesized first using solid-phase peptide synthesis, which is after the cleavage from the polymeric support subjected to oxidation in a buffer system. Redox-active agents such as reduced and oxidized glutathione (GSH/GSSG) are often added to promote the formation of the disulfide bonds. The main disadvantage of buffer-supported self-folding is that the disulfide bonds are not formed selectively in a stepwise fashion. Compared to the native peptide, for which often only one specific disulfide isomer is described, it is possible to obtain numerous other isomers with this approach8. µ-PIIIA has already been shown to result in at least three differently folded isomers upon self-folding in a previous study3. The separation of such an isomer mixture is rather difficult due to similar retention times if using chromatographic purification methods9. The targeted synthesis of a specific isomer is therefore advantageous. To specifically produce an isomer with defined disulfide connectivity, a special strategy is required in which the disulfide bonds are successively closed. Therefore, the linear precursor carrying distinct protecting groups at the individual cysteine pairs is synthesized on the polymer support. After the elimination, the cysteine pairs are individually and successively deprotected and linked in an oxidation reaction to yield the desired disulfide bonds10,11,12,13,14,15,16. After the synthesis and the purification of the reaction product, it is required to confirm the identity and the disulfide connectivity by suitable analytical methods. Numerous analytical methods are available for the elucidation of the primary amino acid sequence, e.g., MS/MS, while the determination of the disulfide connectivity still remains much less investigated. Apart from the complexity of such multiple disulfide-bonded peptides, product-related impurities (e.g., from disulfide scrambling), due to sample preparation and work-up can further complicate analysis. In this paper, we show that the use of a combination of different analytical techniques is necessary to unequivocally clarify the identity of the disulfide bonds in the µ-PIIIA isomers. We have combined chromatographic methods with mass spectrometry and provided the same samples to NMR spectroscopy. In matrix-assisted laser desorption/ionization(MALDI) MS/MS analysis, we identified the disulfide bonds by using partial reduction and iodoacetamide derivatization because top-down analysis is not possible for this peptide. 2D NMR experiments were performed in order to obtain a three-dimensional structure of each isomer. Thus, by combining distinct sophisticated analytical methods, it is possible to elucidate properly the disulfide connectivity and three-dimensional structure of complex multiple disulfide-bonded peptides7.
Note: All amino acids used herein were in the L-configuration. The abbreviations of amino acids and amino acid derivatives were used according to the recommendations of the Nomenclature Committee of IUB and the IUPAC-IUB Joint Commission on Biochemical Nomenclature.
1. Solid-Phase Peptide Synthesis (SPPS)
NOTE: Carry out the synthesis with a solid-phase peptide synthesizer. Perform the synthesis of the linear peptide precursors of the general sequence ZRLCCGFOKSCRSRQCKOHRCC-NH2 using a standard protocol for 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. Apply the following protected amino acids: Pyr(Boc (tert-butyloxycarbonyl)), Arg(Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)), Asn(Trt), Asp(tBu), Hyp(tBu), Lys(Boc), Ser(tBu), Gln(Trt), Glu(tBu), Trp(Boc), Tyr(tBu), Thr(tBu), and His(Trt). Protect the cysteine pairs with Trt-, Acm-, or tBu-groups according to the intended disulfide connectivity.
2. Peptide Cleavage from the Resin (Figure 1A)
NOTE: During the cleavage procedure, all amino acid side chains except for Cys(Acm) and Cys(tBu) will be deprotected. The protocol applies to 100 mg of resin.
3. Purification of the Linear Precursor with Semi-preparative High-performance liquid chromatography (HPLC)
NOTE: Purify the crude peptides by semi-preparative reversed phase (RP) HPLC equipped with a C18 column (5 µm particle size, 100 Å pore size, 250 x 32 mm) and a 3.6 mL injection loop. Use a gradient elution system of 0.1% TFA in H2O (eluent A) and 0.1% TFA in acetonitrile (MeCN)/H2O (9:1, eluent B). Detect the peaks at 220 nm.
4. Selective Formation of the Disulfide Bonds
5. Peptide Purification
NOTE: Purify the oxidized peptides by semi-preparative RP HPLC equipped with a C18 column (10 µm particle size, 300 Å pore size, 250 x 22 mm) and a 3.6 mL injection loop. Use a gradient elution system of 0.1% TFA in H2O (eluent A) and 0.1% TFA in MeCN/H2O (9:1, eluent B). Detect the peaks at 220 nm.
6. Peptide Analytics
7. MS/MS Analysis of Disulfide Connectivity
8. NMR Experiments and Structure Analysis
15 different disulfide-bridged isomers of the µ-conotoxin PIIIA are synthesized and characterized in detail (Figure 1). Disulfide bonds are identified by partial reduction and subsequent MS/MS analysis (Figure 2). NMR analysis of the different isomers is carried out (Figure 3) to reveal the individual peptide structures. Notably, a combination of RP HPLC, MS/MS fragmentation, and NMR analysis is required for unambiguous identification of the disulfide connectivity.
Figure 1: The selective disulfide bond formation of native µ-PIIIA via the protecting group strategy. (A) The deprotection of the Trt-protected cysteines during the peptide cleavage from the resin. (B) The first disulfide bridge formation of the unprotected cysteines. (C) Deprotection and disulfide bridge formation of the Acm protected cysteines. (D) Deprotection and disulfide bridge formation of the tBu protected cysteines. Please click here to view a larger version of this figure.
Figure 2: Partial reduction workflow for the disulfide bond assignment by MS/MS Analysis. (A) Partial reduction and alkylation of the peptide. (B) HPLC purification of different reaction control samples. (C) Reduction of the purified samples. Partially carbamidomethylated species (two peptides in the middle) harbor information on the disulfide connectivity which is determined by MS/MS analysis. This figure has been adapted with permission from Heimer, P. et al. Conformational µ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation. Analytical Chemistry. 90 (5), 3321-3327 (2018). Copyright (2018) American Chemical Society. Please click here to view a larger version of this figure.
Figure 3: The sequential walk and the resulting NMR solution structure of a rigid (left) and a flexible (right) µ-PIIIa isomer. The 20 structures with the lowest energies as well as the disulfide connectivity of the different isomers are shown. The comparison of the root mean square deviation (RMSD) values clarifies that a rigid peptide mostly leads to a better resolved NMR structure. Please click here to view a larger version of this figure.
The method described herein for the synthesis of cysteine-rich peptides such as µ-PIIIA represents a possibility to selectively produce disulfide-bonded isomers from the same amino acid sequence. Therefore, established methods such as Fmoc-based solid phase peptide synthesis18 and a defined protecting group strategy for the regioselective formation of disulfide bonds were used16. The solid-phase peptide synthesis can produce amino acid sequences on a polymer support (resin) via automated synthesis. These amino acids are protected against undesired side reactions during sequence assembly by special protecting groups at their side chains, which are – depending on the protocol used – deprotected upon peptide cleavage from the resin. This protection also applies to the cysteine side chains within the peptide chain, e.g., trityl protection. However, distinct protecting groups for individual pairs of cysteines such as acetamidomethyl and tert-butyl are not concomitantly cleaved through this elimination step. These protecting groups can be cleaved off under conditions which allow subsequent oxidation by forming disulfide bonds from the deprotected thiol groups. In this way, all 15 disulfide isomers of a peptide containing six cysteine residues can be selectively formed7,16. The limiting factor, however, is that with an increasing number of different orthogonal protecting groups the synthetic effort increases, which has a high impact on the yield of the desired disulfide bridged peptide. Thus, in selected cases and in particular for less complex peptides possessing only one or two disulfide bonds the oxidative self-folding approach indeed might be preferred over the protecting group strategy.
Herein, the Trt groups of one cysteine pair are removed by the action of TFA after completion of the peptide assembly and final Fmoc-deprotection of the N-terminal amino acid. The acidic conditions, however, leave the Acm and tBu protecting groups at the other two pairs of cysteine residues intact. Subsequently, purification of the linear peptide containing two unprotected cysteines is performed using preparative HPLC under slightly acidic conditions to maintain the thiol groups in the protonated form. The protocol continues with the first oxidation step after analytical HPLC and MS analysis verified successful synthesis and deprotection of Trt at the cysteine pair of interest. The further steps of deprotection and oxidation of the second and third disulfide bond are carried out and confirmed in the same way using the appropriate protocol for Acm and tBu, respectively. These oxidation reactions are thus simple wet-chemical reactions performed in solution which do not require expensive reagents. The disadvantage, however, is that certain reactions, i.e., connections of distinct cysteine residues, do not proceed smoothly and completely leading to by-products of scrambled disulfide connectivity. Since these are not removed after completion of the individual oxidation reactions, such by-products can accumulate in the crude product mixture. Some of these may possess similar physicochemical properties as the actual peptide, e.g., HPLC elution, which increases the effort for purification of the correctly folded peptide. Although the synthesis and purification could be difficult, as is the case for µ-PIIIA, this method can successfully be used, yet requires good manual and observation skills. Finally, it needs to be considered that every peptide sequence is different and therefore might be treated differently in order to be successful in generating the correct disulfide connectivity.
In addition to the challenging synthesis, it is essential to verify whether the disulfide bonds produced are correct, i.e., represent the intended version of the respective disulfide isomer. This is performed herein using a combination of MALDI MS/MS analysis and NMR structure elucidation. MS/MS analysis is carried out using different partially reduced and alkylated derivatives (carbamidomethylation) of the fully oxidized peptide17. In the MALDI MS/MS LID TOF/TOF spectra an even number of carbamidomethylated cysteines is always found, i.e., 2-, 4- or 6-times carbamidomethylated. This can be explained by the stepwise reduction of the completely oxidized peptides, since in each occasion two thiol functions per disulfide bond are always reduced (opened) and in situ alkylated using iodoacetamide. This carbamidomethylation of two cysteines each can be detected in the MALDI MS/MS spectra and, in turn, refers to the respective disulfide bond these cysteines were engaged in in the intact peptide. For example, the occurrence of four carbamidomethylated cysteines in a sequence with three disulfide bonds helps to identify one specific bond, namely the one formed between the two non-alkylated cysteines, which were not opened during the reaction time of the partial reduction sufficient to open the other two bonds. Thus, by MALDI MS/MS analysis of the various 2- and 4-times carbamidomethylated derivatives of the peptide disulfide isomer, the disulfide connectivities can be completely elucidated and confirmed.
Another possibility to elucidate the disulfide bond pattern is the MS/MS analysis of enzymatically digested peptides, where the peptide has to be proteolytically cleaved at distinct amino acids to produce smaller fragments. These short fragments can still be linked via disulfide bonds, which is the reason that the disulfide pattern can be elucidated from MS/MS analysis of these linked fragments. In case of µ-PIIIA, however, this strategy could not be applied because some cysteines of the sequence are directly adjacent to each other and therefore the digestion of the peptides does not separate the cysteines from each other. Identification of the specific disulfide bridge is therefore difficult.
The structure elucidation by 2D NMR analysis is clearly facilitated in case of a known disulfide connectivity, because this knowledge enables the assignment of nuclear Overhauser effect (NOE's) to specific protons, i.e., referring to the spatial distance between two atoms determined from the intensities of the NOESY signals (through-space NMR experiment). The analysis, however, starts with the sequential walk19 applied to the COSY, TOCSY and NOESY spectra, in this way, it is possible to assign a specific signal to the corresponding H-atom of the amino acids (spin system) in the sequence. The aforementioned distance restraints from the NOESY experiment are used in the structure calculation of the peptide7. The more signals identified, the more accurate the structure will be. However, the difficulty of this assignment increases with the number of amino acids in the peptide and with the occurrence of adjacent cysteines, as the probability increases that signals of several atoms overlap and can no longer be precisely assigned due to close proximity. Furthermore, the flexibility of the peptide chain is decisive for whether signals in the NMR are easily or hardly identifiable. The more flexible a peptide region is, the more conformational changes are occurring, allowing multiple signals to be obtained for one and the same atom. Thus, the intensity decreases with the number of conformations, which causes signals to disappear into background noise. This means that three-dimensional structure elucidation via NMR becomes much easier if the sequence is conformationally constrained.
Finally, this protocol makes it possible to generate multiple disulfide-bridged peptides by monitoring the disulfide bond formation by MALDI MS/MS analysis and concomitant 2D NMR structure elucidation7.
The authors have nothing to disclose.
We would like to thank A. Resemann, F. J. Mayer, and D. Suckau from Bruker Daltonics GmbH Bremen; D. Tietze, A. A. Tietze, V. Schmidts and C. Thiele from the Darmstadt University of Technology; O. Ohlenschläger from the FLI Jena, M. Engeser from the University of Bonn; K. Kramer, A. Harzen, and H. Nakagami from the Max Planck Institute for Plant Breeding Research, Cologne; Susanne Neupert from the Institute for Zoology, Cologne; and the Biomolecular Magnetic Resonance Spectroscopy Facilities of the University of Frankfurt for technical support, training modules, and access to instruments. Financial support by the University of Bonn to D.I. is gratefully acknowledged.
Fmoc Rink amide resin | Novabiochem | 855001 | |
Pyr(Boc) | Bachem | A-3850 | |
Arg(Pbf) | Iris Biotech | FSC1010 | |
Asn(Trt) | Bachem | B-1785 | |
Asp(tBu) | Iris Biotech | FSP1020 | |
Hyp(tBu) | Iris Biotech | FAA1627 | |
Lys(Boc) | Bachem | B-1080 | |
Ser(tBu) | Iris Biotech | FSC1190 | |
Gln(Trt) | Iris Biotech | FSC1043 | |
Glu(tBu) | Iris Biotech | FSP1045 | |
Trp(Boc) | Iris Biotech | FSC1225 | |
Tyr(tBu) | Sigma Aldrich | 47623 | |
Thr(tBu) | Iris Biotech | FSP1210 | |
His(Trt) | Iris Biotech | FDP1200 | |
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorphosphat | Sigma Aldrich | 8510060 | Flammable |
DMF | Fisher Scientific | D119 | Flammable, Toxic |
DCM | Fisher Scientific | D37 | Carcinogenic |
Piperidine | Alfa Aesar | A12442 | Flammable, Toxic, Corrosive |
N-Methyl-Morpholin | Sigma Aldrich | 224286 | |
Cys(Acm) | Iris Biotech | FAA1506 | |
Cys(Trt) | Bachem | E-2495 | |
Cys(tBu) | Bachem | B-1220 | |
trifluoruacetic acid | Sigma Aldrich | 74564 | Toxic, Corrosive |
phenol | Merck | 1002060 | Toxic |
thioanisol | Alfa Aesar | A14846 | |
ethanedithiol | Fluka Analytical | 2390 | |
diethyl ether | VWR | 100,921 | Flammable |
tert-butanol | Alfa Aesar | L12338 | Flammable |
acetonitrile | Fisher Scientific | A998 | Flammable |
water | Fisher Scientific | W5 | |
isopropanol | VWR | ACRO42383 | Flammable |
sodium hydroxide | AppliChem | A6579,1000 | Corrosive |
iodoacetamide | Sigma Aldrich | I6125 | |
iodine | Sigma Aldrich | I0385 | |
Hydrochloric acid | Merck | 110165 | Corrosive |
ascorbic acid | Sigma Aldrich | A4403 | |
diphenylsulfoxide | Sigma Aldrich | P35405 | |
anisol | Sigma Aldrich | 96109 | Flammable |
trichloromethylsilane | Sigma Aldrich | M85301 | Flammable |
sample dilution buffer | Laborservice Onken | ||
sodium dihydrogen phosphate | Sigma Aldrich | 106370 | |
disodium hydrogen phosphate | Sigma Aldrich | 795410 | |
(2-carboxyethyl)phosphine hydrochloride | Sigma Aldrich | C4706 | |
citric acid | Sigma Aldrich | 251275 | |
sodium citrate dihydrate | Sigma Aldrich | W302600 | |
tris-acetate | Carl Roth, | 7125 | |
Ethylenediaminetetraacetic acid | Sigma Aldrich | E26282 | |
peptide calibration standard II | Bruker Daltonics GmbH | 8222570 | |
Name of Equipment | Company | ||
solid-phase peptide synthesizer | Intavis Bioanalytical Instruments AG | EPS 221 | |
lyophilizer | Martin Christ GmbH | Alpha 1-2 Ldplus | |
semipreparative HPLC | Jasco | system PV-987 | |
Eurospher 100 C18 column (RP, 5 µm particle size, 100 Å pore size, 250 x 32 mm) | Knauer | 25QE181E2J | purification of the linear peptide |
Vydac 218TP1022 column (RP C18, 10 µm particle size, 300 Å pore size, 250 x 22 mm) | Hichrom-VWR | HICH218TP1022 | purification of the oxidized peptide |
analytical HPLC | Shimadzu | system LC-20AD | |
Vydac 218TP54 column (C18 RP, 5 µm particle size, 300 Å pore size, 250 x 4.6 mm) | Hichrom-VWR | HICH218TP54 | analytical column |
ground steel target (MTP 384) | Bruker Daltonics GmbH | NC0910436 | MALDI preparation |
C18-concentration filter (ZipTip) | Merck KGaA | ZTC18S096 | MALDI preparation |
MALDI mass spectrometer | Bruker Daltonics GmbH | ultraflex III TOF/TOF | |
amino acid analyzer | Eppendorf-Biotronik GmbH | LC 3000 system | |
NMR spectrometer Bruker Avance III | Bruker Daltonics GmbH | Bruker Avance III 600 MHz | |
computer program for molecular visualising | YASARA Biosciences GmbH | Yasara structures | NMR structure calculation |
computer program for MALDI data evaluation | Bruker Daltonics GmbH | flexAnalysis, BioTools | MS/MS fragmentation |
analog vortex mixer | VWR | VM 3000 | |
Microcentrifuge | Eppendorf | 5410 | |
Centrifuge | Hettich | EBA 20 | |
Rotational vacuum concentrator | Christ | 2-18 Cdplus | |
Analytical Balance | A&D Instruments | GR-202-EC |