Efficient Primary Structure Elucidation of Disulfide-bridged Peptides
The combination of Edman sequence analysis and electrospray tandem mass spectrometry provides accurate and detailed structural data of highlyknotted spider toxins
The primary structure determination of disulfide bridged peptides is greatly facilitated using a combination of the Procise® cLC 492 sequencing system and the QSTAR® Pulsar hybrid quadrupole time-of-flight mass spectrometer. These two techniques provide complementary data for an unambiguous characterization of peptides and proteins, while simultaneously extending the range of applications.
N-terminal amino acid sequence analysis by Edman degradation has been the workhorse for the de novo sequence determination of peptides and proteins for many years, especially since the introduction of an automated sequenator in 1967 by Per Edman himself. Modern sequencing systems based on the Edman chemistry provide high sensitivity and enable fully automated analyses. Due to the unambiguous sequence data generated, Edman sequencing is still the reference technology for complete de novo protein sequencing. As a further key technology, mass spectrometry has entered the field of bioanalysis within the last decade, enabling rapid protein identification either by peptide mass mapping or by determination of partial amino acid sequences. Besides that, the impressive resolving power and mass accuracy of modern mass spectrometric instrumentation offers a great potential for elucidation of structural details. The combination of Edman sequencing and tandem mass spectrometry is an efficient approach to the primary structure elucidation of complex, highly disulfide-bridged spider toxins.
• Total de novo sequencing approach
• Fully automated amino acid sequence determination using the Procise cLC sequencing system
• Highly accurate electrospray tandem mass spectrometry for the elucidation of disulfide-bridge patterns using the QSTAR® Pulsar hybrid quadrupole time-of-flight mass spectrometer
• Combination of two highly senisitive technologies provides information on complex disulfide structures
Sample Preparation: The multicomponent venom of the spider Cupiennius salei (Figure 1) was purified by a combination of gel filtration, cation exchange chromatography and reversephase HPLC1.
Edman sequencing: Two important neurotoxic peptides1,2,3, CSTX-1 (8'351.90 Da) and CSTX-9 (7'530.25 Da) were subjected to sequence determination by Edman degradation in a Procise cLC 492 protein sequencer. The reduced Efficient Primary Structure Elucidation of Disulfide-bridged Peptides Application Note Peptide Sequencing Tandem Mass Spectrometry www.appliedbiosystems.com Figure 1. Venom from Cupiennius salei was used in this study. and alkylated samples were sequenced up to Ile 53 (CSTX-1) and Ala 47 (CSTX-9), respectively. In order to secure the rest of the sequences, reduced and alkylated CSTX-1 was cleaved with endoproteinase Asp-N and chymotrypsin, CSTX-9 with endoproteinase Asp-N and immobilised trypsin. In each case the peptides generated were separated by RP-HPLC and identified by amino acid analysis and mass spectrometry. Native CSTX-1 and CSTX-9 were cleaved with immobilised trypsin to eliminate disulfide exchange during digestion, and iodoacetamide was added as a scavenger to alkylate any free thiol groups that may have been formed during cleavage. The tryptic peptides were separated by RP-HPLC and the disulfide-containing peptides were identified by amino acid analysis and by tandem mass spectrometry. Electrospray tandem mass spectrometry: Identification of the disulfide-bridge pattern was performed by nano-electrospray tandem mass spectrometry on the QSTAR® Pulsar hybrid quadrupole time-of-flight mass spectrometer2, 4. The purified native peptides were digested by trypsin and Asp-N endopeptidase. The resulting peptides consisted of four to five chains crosslinked by the original disulfide bridges. The peptides were subsequently subjected to MS and MS/MS analysis. Lyophylized samples were redissolved in water/methanol (50/50) and an aliquot of water/methanol/formic acid (49/49/2) was added prior to analysis, resulting in a final peptide concentration of 5 picomoles per microliter. The instrument was tuned for a resolving power of 12'000, which provided sufficient separation of fragment ions of similar m/z and unambiguous assignment of charge states.
Results and Discussion
The complete sequences of CSTX-1 and CSTX-9 with the corresponding overlaps are given in Figure 2. Sequence comparison of CSTX-9 with CSTX-1 revealed an identity of 53% as well as identical positions of all eight Cys residues present in the N-terminal portion of the molecule. Sequence comparison with other spider toxins (Figure 3) is indicative for a disulfide bridge pattern as in the family of ion channel toxins containing the inhibitor cystine knot structural motif. The proximity of the cysteine residues and the absence of suitable cleavage sites within the amino-acid sequences of CSTXpeptides caused the classical approaches for elucidation of the disulfide bridge pattern to fail. As these approaches are based on selective chemical or enzymatic cleavage, they require the presence of defined cleavage sites between two consecutive disulfide bridges. Consequently, the basically unspecific gas-phase dissociation of the disulfide-linked peptides by tandem mass spectrometry is an attractive alternative to the classical procedures. Identification of the disulfide bridge pattern was based on observation of characteristic fragment ions generated by dissociation of the peptidic bonds adjacent to the cysteines, as illustrated in Figure 3. The identification of the disulfidebridge pattern of the CSTX peptides by tandem mass spectrometry represents a valuable alternative to traditional methods for elucidation of complex molecular structures. Modern highperformance instru- mentation, such as the QSTAR® Pulsar mass spectrometer, greatly enhance the potential of this technique, since a wide range of information is obtained from a single experiment using minute amounts of sample only. Sample preparation is essentially reduced to a purification step, thus, avoiding the risk of potential structural alteration, e.g. disulfide scrambling.
CSTX-1 and CSTX-9 share the same disulfide bridge pattern linking Cys 1 – Cys 4, Cys 2 – Cys 5, Cys 3 – Cys 8, Cys 6 – Cys 7. This pattern is also found in other spider toxins, e.g. w-agatoxins and μ-agat oxins from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins from Hololena curta (Figure 3). This disulfide bridge pattern represents thewell-known consensus sequence of the inhibitor cystine knot structural motif: CX3-7CX3-10CX0-7CX1-8CX4-20C. The results demonstrate that Edman sequencing and tandem mass spectrometry are by no means competing techniques. These two techniques can be considered as complementary tools, each one offering its particular advantages. Edman sequencing is a straight forward and efficient approach to the determination of amino acid sequences of peptides and proteins, whereas tandem mass spectrometry exhibits its ultimate potential for the elucidation of structural details. If both techniques are available to the researcher, bioanalytical problems are solved most accurately and efficiently.
1L. Kuhn-Nentwig, J. Schaller, W. Nentwig, Purification of toxic peptides and the amino acid sequence of CSTX-1 from the multicomponent venom of Cupiennius salei (araneae: ctenidae). Toxicon, 1994, 32, 287-302. 2J. Schaller, U. Kämpfer, S. Schürch, L. Kuhn- Nentwig, S. Haeberli, W. Nentwig, CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulfide bridge pattern and comparison with other spider toxins containing the cystine knot structure. Cell Mol. Life Sci., 2001, 58, 1538-1545. 3J. Schaller, L. Kuhn-Nentwig, S. Schürch, U. Kämpfer, J. Müller, W. Nentwig, Neurotoxic peptides in the multicomponent venom of the spider Cupiennius salei. Part I. Primary structure of neurotoxic peptides in relation to their biological function. Chimia 2001, 55, 1058-1062. 4S. Schürch, J. Schaller, U. Kämpfer, L. Kuhn- Nentwig, W. Nentwig, Neurotoxic peptides in the multicomponent venom of the spider Cupiennius salei. Part II. Elucidation of the disulfide-bridge pattern of the neurotoxic peptide CSTX-9 by tandem mass spectrometry. Chimia 2001, 55, 1063-1066.
Stefan Schürch1, Johann Schaller 1, Urs Kämpfer 1, Lucia Kuhn-Nentwig2, Stefan König3 1Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland 2Zoological Institute, University of Bern, Baltzerstrasse 6, CH-3012 Bern, Switzerland 3Applied Biosystems, Grundstrasse 10, 6343 Rotkreuz, Switzerland