Electron transfer dissociation coupled to an Orbitrap analyzer may promise a straightforward and accurate sequencing of disulfide-bridged cyclic peptides: a case study.

Dear Sir, Electron transfer dissociation coupled to an Orbitrap analyzer may promise a straightforward and accurate sequencing of disulfide-bridged cyclic peptides: a case study Cyclic peptides constitute a large group of compounds that possess potent biological and pharmacological activities, and thus have attracted ever-growing attentions in areas such as drug development and biomedical research [1]. Therefore, the ability to analyze cyclic peptides by high-throughput methods such as the tandem mass spectrometry (MS) will be of high interest [2]. Nevertheless, sequencing of these peptides by conventional MS techniques, which typically employ collision induced dissociation (CID) to obtain sequence-informative fragments, represents a daunting challenge. Cyclic peptides maintain a highly rigid conformation due to a circular backbone, which often accounts for their high receptor selectivity, metabolic stability, and potency [1]. Circular backbones present several challenges when sequencing by CID. First, the largely random initial ring scissions of the backbone often result in a perplexing mixture of different sets of b/y ions and neutral loss/internal fragments [3], rendering the CID spectra very complex [2, 4]. Second, the CID fragmentation of cyclic peptides often produces many fragments that are not typical for the cleavage of a linear peptide (such as sequence scrambling product ions), and thus are difficult to interpret [4, 5]. Third, the cyclic backbone of a majority of therapeutic cyclic peptides is held together by a disulfide bond that cannot be cleaved efficiently utilizing the CID mechanism [6]. Consequently, the application of CID for the structure elucidation of cyclic peptides is severely limited. Electron transfer dissociation (ETD), which is an analogue of the electron capture dissociation (ECD) technique, converts multiple-charged peptide cations into radicals and cleaves the N-Cα bonds, providing a fragmentation mechanism orthogonal to that by CID [7, 8]. The resulting fragment ions that contain the N- and C- termini of the peptides are termed c and z ions, respectively. It has been demonstrated that the electron-initiated dissociation during an ETD process is less discriminated by the amino acid residues and side-chain chemistry compared with CID [7]. As a result, in most cases ETD provides more complete peptide backbone cleavage, and shows particular strength in the identification of post-translational modifications [7, 8]. Another advantage of ETD over CID is that it provides efficient cleavage of disulfide bonds (cf. the discussion), which may enable the localization of disulfide linkages in peptides or intact proteins under non-reducing conditions. Therefore, it is possible to utilize the ETD technique to achieve a favorable sequencing of the disulfide-bridged cyclic peptides, which account for most of the cyclic peptides. So far only a few studies have been conducted on the fragmentation of cyclic peptides by ETD or ECD [9, 10]. As a result, a detailed investigation of the ETD behaviors of cyclic peptides, as well as an evaluation of the effectiveness of ETD for sequencing these peptides, is highly valuable. Moreover, in-depth understanding of the underlying mechanisms of ETD reactions on cyclic peptides may contribute to our knowledge of this relatively new technique. In this report, we studied the ETD fragmentation of the model cyclic peptide eptifibatide, which is a potent antagonist for glycoprotein IIb/IIIa and has been widely used in clinical practices to inhibit platelet aggregation [11]. Our study demonstrated CID of this disulfide-containing cyclic peptide failed to produce a sequence-informative fragmentation pattern, confirming the previous observations [11, 12]. Here we hypothesized that ETD would likely make an initial ring scission on the disulfide that holds together the circular backbone, resulting in a more simplified and sequence-informative spectra when compared to CID. This hypothesis was tested by performing both CID and ETD on eptifibatide and comparing the spectra of the activation methods. In order to ensure highly confident identifications of the fragments, a high resolution and high mass-accuracy Orbitrap was employed. Eptifibatide (purity > 99%), , where Mpr is 3-mercaptopropionic acid and Har is homoarginine, was kindly provided by Merck/Schering-Plough (North Wales, PA). The standard solution (100 ng/mL, freshly prepared in 2% ACN) was loaded onto the trap column (Zorbax Stablebond-C8, 5×0.3 mm I.D., Agilent, Santa Clara, CA), via an Eksigent AS2 autosampler (Eksigent Technologies, Dublin, CA). The injection volume was 4 μL. The analytical column is a 150 mm×75 μm I.D nano column packed with Pepmap C18 material. In order to minimize potential ion suppression or interferences from other components in the sample, a relatively shallow nanoflow gradient was adopted from our previous work [13]. The mobile phases were delivered by four Eksigent direct-flow capillary/nano-LC pumps (Dublin, CA). Mass spectrometry analysis was performed on a hybrid linear LTQ/Orbitrap mass spectrometer with ETD (Thermo Electron, CA). In order to ensure the mass accuracy for this study, the Oribtrap was calibrated on the same day that the experiments were conducted. The instrument was operated under data-dependent mode to fragment the most abundant precursors with alternating ETD and CID. The resolution of the Orbitrap was set at 60000 for both full scan and product ion scan. The target value for MS/MS in the Orbitrap is 2×105 and target value for fluoranthene anions is 1×106. As shown in SI Fig 1, the full scan spectrum of eptifibatide under the current LC/MS conditions was dominated by singly- and doubly-protonated ions. Both electron transfer and proton transfer processes were observed in the ETD fragmentation of the doubly charged eptifibatide, generating a charge-reduced ion at m/z 833.329 and a proton-deprived ion at m/z 832.324 respectively. Because the proton-deprived product hardly fragment further [7], the strewed product ions in the ETD spectrum (Fig. 1) were mostly from the further dissociation of the charge-reduced ion [M+2H]+· (m/z 833.329). These product ions can be putatively categorized into three groups: i) typical backbone cleavage; ii) side-chain cleavage and small neutral loss; and iii) conformation-dependent fragmentation. Detailed interpretations for each category are shown below. In order to ensure the accuracy of interpretation, all assignments required a mass error < 5 ppm. The calculation of the degree of unsaturation (i.e. rings-plus-double bonds, RDB), was employed to determine whether the fragment ions/neutral loss moieties carry even or odd electrons (Tab 1). Figure 1 Production scan spectrum of doubly protonated eptifibatide following ETD. The data were acquired by an Orbitrap mass spectrometry with the resolution set at 60 000. In addition to backbone ions, fragments resulting from side chain cleavage are also observed ... Table 1 High resolution MS analysis of the fragments from doubly protonated eptifibatide following ETD