Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation
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- Mormann, M., Eble, J., Schwöppe, C. et al. Anal Bioanal Chem (2008) 392: 831. doi:10.1007/s00216-008-2258-7
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Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.
KeywordsInter-and intramolecular disulfide bridgeUnderivatised peptidesLow-energy collision-induced dissociationAsymmetric disulfide-bridge cleavage
Oxidative coupling of the reactive SH groups of cysteines, yielding disulfide bonds, is one of the most important processes in the formation of the native structure of proteins. Often, disulfide bridges enhance the stabilisation of the tertiary structure of folded proteins [1–4].
Classical approaches for disulfide bond determination involve chemical or enzymatic cleavage of the intact protein followed by separation of the resulting peptides. Disulfide-linked peptides are then identified by rupture of the S-S bond, thereby modifying the separation properties. The latter are monitored by either electrophoretic or chromatographic techniques [5–7].
More recently, mass spectrometric techniques have been used for analysis of disulfide-linked proteins and peptides [8, 9]. Identification and sequencing of proteins is most frequently performed by use of proteolytic digestion followed by mass spectrometric analyses. Standard procedures for enzymatic degradation usually involve reductive cleavage of disulfide bonds present in the protein, to disrupt its tertiary structure. Reductive cleavage of the disulfide bonds is then followed by alkylation of the reactive free sulfhydryl groups preventing spontaneous oxidation by other sulfhydryl groups. However, when this approach is used, information on covalent cysteine-cysteine contacts within the protein is lost.
Various approaches have been shown to result in valuable information on analyte species in which two peptides are linked by a disulfide bridge. Cleavage of the disulfide bonds has been observed in both long-lived metastable ions and in ions fragmenting on a much shorter time-scale in the ion source when gas-phase ions are generated by matrix-assisted laser desorption/ionisation [10–17]. Along with either symmetric or unsymmetric fragmentation of the S-S bridge, cleavage of amide bonds is often observed, giving rise to (partial) sequence information on the peptide ions. Electron-capture dissociation (ECD) is an alternative approach to the analysis of disulfide or sulfide-containing species, because it has been shown that C-S and S-S bonds are cleaved extremely rapidly and easily in hypervalent radical cations formed upon capture of an electron by a multiply charged precursor ion species [18–23].
Defining the linkage pattern and obtaining sequence information from peptide ions containing an intramolecular disulfide loop by low energy collision-induced dissociation (CID) still remains a somewhat elusive goal. Peptide bonds within the disulfide loop are reported to be usually resistant to fragmentation under low-energy CID conditions which are typically applied to de-novo sequencing of peptides . This finding is corroborated by recent results obtained by Lioe and O’Hair . They examined the fragmentation of protonated model compounds containing intermolecular disulfide links both experimentally and theoretically. The results indicate that most cleavages of the disulfide bridge proceed usually via dissociation pathways that cannot compete with amide bond cleavages energetically. However, especially when peptide ions containing non-mobile protons are considered, disulfide bond cleavage reactions might be observed .
In some cases this problem can be overcome by use of high-energy collisional activation leading to both cleavage of the disulfide bond and fragmentation of the amide bonds of the peptide backbone . However, the high-energy regime is not accessible with most instruments commonly used in proteomics laboratories. Complexation with gold(I) ions and other transition metals is an alternative approach to disulfide link cleavage, owing to the high sulfur affinity of heavier transition metals [26–28]. Kim and Beauchamp have reported a novel method for identifying disulfide linkages in peptides by use of multiple-stage CID of analyte species cationised with alkali and alkaline earth metal ions . Recently, Thakur and Balaram have reported on the fragmentation of positively charged gas-phase ions generated from contryphans, peptides containing a single disulfide bridge . It has been reported that these analyte species mainly fragment by initial cleavage of an amide bond within the disulfide loop, giving rise to a linearised sequence. This process is promoted by the presence of at least one proline residue within the S-S loop, because amide bond cleavage adjacent to proline is a fast and facile process . Ring-opening is typically succeeded by several dissociation processes giving rise to valuable information about the species under investigation. However, if no proline residues are present within the peptide sequence fragmentation by linearisation is rarely observed.
In this contribution we present recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation (nanoESI CID). Careful examination of the fragment ion spectra revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge, giving rise to a modified cysteine-containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.
Reagents and materials
Rhodocetin alpha subunit from the venom of the Malayan pit viper (Calloselasma rhodostoma) was obtained by isolation and purification as described before . A modified fusion protein comprising the extracellular domain of the tissue factor fused to the peptide GNGRAHA (tTF) was expressed in E. coli, isolated and purified according to the procedure reported by Kessler et al. . Trypsin was purchased from Roche Diagnostics (Mannheim, Germany). All solvents were of HPLC-grade purity.
Omega glass capillaries used in nanoESI experiments were purchased from Hilgenberg (Malsfeld, Germany), and pulled by use of an in-house-built vertical pipette puller.
Proteins were dissolved in 10 mmol L−1 ammonium hydrogen carbonate to final concentrations of 10 pmol μL−1 and first partially denatured by thermal defolding (95 °C, 5 min). Treatment of 10 μL protein solution with trypsin (20 μg mL−1) in solution (NH4HCO3, 10 mmol L−1, pH 7) for 12 h at 37 °C furnished the peptide mixtures analysed in this study. The mixtures were dried in a Speedvac (Savant, Farmingdale, NY, USA), and the dried residue was dissolved in 10 μL water to yield a concentration of 10 pmol μL−1 for the stock solution.
Mass spectrometric measurements
Nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry
Measurements were performed using a Bruker Apex II Fourier-transform ion-cyclotron resonance mass spectrometer (FT-ICR MS) equipped with a 9.4 T actively shielded magnet. Gas-phase ions were generated from solutions containing approx. 7.5 pmol μL−1 of the analyte material in water-methanol-formic acid 68:22:10 (v/v/v) by nanoESI in the positive-ion mode using an Apollo ion source. Typical source conditions were: capillary voltage −650 V and a capillary exit voltage of 60 V. The electrospray-generated ions were accumulated for 0.5 s in the hexapole located after the 2nd skimmer of the ion source and then transferred into the ICR cell. The ions were trapped inside the Infinity ICR cell by application of a “sidekick”, the trapping voltages were set to +0.9 V at both trapping electrodes. All mass spectra were acquired in the broadband mode with 512 kword data points. The time-domain signals were zerofilled once and apodized by a quadratic sine bell function prior to Fourier transformation. For all spectra 64 scans were accumulated.
Spectra were calibrated internally by use of the characteristic peptide ions generated by auto-digestion of trypsin.
Nanoelectrospray quadrupole time-of-flight tandem mass spectrometry (nanoESI-Q-TOF MS-MS)
Results and discussion
Peptide mixtures obtained from proteolytic digests of partially denatured proteins, achieved by thermal defolding, where first investigated by high-resolution Fourier-transform ion-cyclotron resonance mass spectrometry (FT-ICR MS). Owing to the high mass accuracy of the instrument used oxidised species, i.e., those with a disulfide bridge, were readily identified by their exact mass (cf. Table 1).
Charge-induced cleavage of the disulfide bridge, i.e., protonation of S-S as the initial fragmentation step, is improbable, because of the low proton affinity of the disulfide bridge [18, 37]. Therefore, a charge-remote intramolecular proton abstraction from the α-position of a cysteine residue by a nucleophilic and highly basic functional group present within the peptide leads to ring opening of the disulfide loop. Then, proton transfer to the disulfide moiety gives rise to a disulfohydryl substituent and a dehydroalanine residue at the C-S cleavage site of the former disulfide loop. The results obtained by Lioe and O’Hair indicate that the activation barrier for such a “salt bridge mechanism” is sufficiently low (145 kJ mol−1) to compete with the dissociation of the amide bonds outside and inside the loop (100–165 kJ mol−1). This process is followed by rupture of the amide bonds present in the peptide ions leading to b and y-type ions with the respective mass shift. For proper annotation of the resulting fragment ions those species containing the Dha amino acid residue have been denoted α-type ions and those containing the S-SH modification were denoted β-type ions.
To further investigate whether this type of cleavage can be regarded as a general fragmentation process of disulfide-bridge-containing peptides under low-energy CID conditions tryptic fragments obtained from a modified truncated tissue factor (tTF) have also been submitted to MS-MS experiments.
Figure 3 shows a nanoESI Q-TOF fragment ion spectrum obtained from a CID experiment with the doubly charged precursor ions with m/z 997.49 [PC + 2H]2+. A similar fragmentation pattern as observed for peptide ions [PA + 3H]3+ and [PB + 3H]3+ is detected. Cleavage of the intramolecular disulfide bridge giving rise to formation of a dhA residue at the C-terminal cysteine residue and a thiocysteine at the cysteine residue located closer to the N-terminus is followed by formation of b and y-type ions. Even if the resulting fragment ions give rise to rather weak signals these data can provide almost the complete peptide sequence assignment.
The peptide PD represents the amino acids 1 to 18 and the peptide backbone is cleaved at K10. The peptides are referred to as P1 (aa 1–10) and P2 (aa 11–18) and b and y-type ions are prefixed with the corresponding numbers. Again, complete sets of y-type ions and a number of b-type ions can be detected and allow the determination of the peptide sequences and location of the disulfide bridge. Moreover, fairly abundant ions derived from α and β-fragmentations of the disulfide bond and from their combinations with b and y-type cleavages can be found. However, in contrast to intra-peptide disulfide bonds no preferred β-cleavage in combination with y-type ions is found. This observation is made also in the CID spectra of other peptides containing intermolecular disulfide bridges (e.g., chymotryptic peptide ions derived from the rhodocetin alpha subunit; data not shown). This finding might indicate that asymmetric cleavage of the intermolecular disulfide bridges does not necessarily precede amide bond fragmentation.
asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine (dhA) residue on the C-S cleavage site; and
fragmentation of the amide bonds giving rise to modified b and y-type ions shifted by −34 u and +32 u, respectively.
This type of fragmentation can be used for characterisation of proteins containing disulfide bridges with respect to both de-novo sequencing and disulfide bridge location.
We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 492, project Z2 to JPK).