Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides
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A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.
KeywordsCyclic peptide Stapled peptide Ornithine effect
Cyclic, stapled, and branched peptides constitute a unique and growing class of biomolecules with promise as therapeutics because of their biostability and resistance to proteolytic digestion in physiological environments [1, 2, 3, 4, 5, 6, 7]. In the context of therapeutics, peptide-based drug candidates display both advantages and disadvantages in comparison to their small molecule counterparts. Peptides, while frequently less toxic than small molecules when administered intravenously and exhibiting higher selectivity for specific biological functions, do not traverse cell membranes with the ease that small molecules do and are prone to proteolytic degradation [8, 9]. However, peptides that are protected from degradation via cyclization or stapling have been shown to exhibit high potency and low toxicity, resulting in more promising candidates for drug administration than their linear counterparts [3, 4, 5, 6, 7]. A myriad of nonlinear peptides are found naturally in plants, fungi, and bacteria as well as synthetic ones produced in the laboratory [10, 11, 12, 13, 14, 15, 16, 17, 18]. Valinomycin, for example, is a cyclic dodecadepsipeptide produced by Streptomyces fulvissimus with potent antibacterial properties while also acting as a potassium-selective ionophore [19, 20]. Enzyme inhibition is another pharmaceutical application that is associated with cyclic peptides, as shown by two examples of the Bowman-Birk class of protease inhibitors, Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) .
From an analytical standpoint, structural characterization of cyclic or stapled peptides is significantly more challenging than elucidation of linear peptides. The success of tandem mass spectrometry for sequencing peptides is based on production of predictable N-terminus and C-terminus fragment ions via cleavage of the peptide backbone. Cyclic peptides require cleavage of two backbone bonds to generate fragment ions, and the lack of natural N-terminal and C-terminal positions confounds an orderly mapping of the sequence of residues. Stapled peptides suffer from a similar pitfall in the region of the peptide containing the stapled (cyclized) portion and also typically contain hydrocarbon (non-peptide-like) linkers that constitute the staple. Several mass spectrometric techniques, including collision induced dissociation (CID) [20, 21, 22, 23, 24, 25], MSn methods [23, 25, 26, 27], electron capture dissociation (ECD) , and complexation strategies [29, 30], have emerged as the most valuable tools to assist in the characterization of the sequences of non-linear peptides.
In addition to the use of MSn methods to facilitate the characterization of cyclic and stapled peptides, another synergistic strategy evolves from site-selective fragmentation processes that may be used to “anchor” a particular location in a molecule based on a site-specific cleavage. Once an anchor point is established, all other fragmentation pathways and the resulting product ions can be referenced to the anchor point. Highly selective or preferential bond-specific cleavages remain relatively rare occurrences upon activation of peptides and proteins. As one example, the proline effect is one of the few well-established site-specific cleavage upon activation, occurring N-terminal to proline residues due to the increased basicity of the N-alkylated amide bond as well as the increased steric hindrance about the residue [31, 32]. The proline-directed cleavage creates a readily recognized fragment ion in the MS/MS spectra of peptides. Along these lines, there has been renewed interest in the development of peptide derivatization strategies to install tags with labile bonds or ones with selectively cleavable groups in order to promote bond-selective cleavages [33, 34, 35, 36, 37, 38, 39]. Recently, the “ornithine effect” has been reported as a site-specific cleavage occurring C-terminal to ornithine residues [40, 41]. Upon collisional activation of an ornithine-containing peptide, the amine of the ornithine residue cyclizes via nucleophilic attack at the adjacent carbonyl group, resulting in a characteristic and preferential cleavage C-terminal to the carbonyl group. This phenomenon has been observed before, as lysine and many of its homologues have exhibited similar characteristics as a nucleophile previously [42, 43, 44, 45].
The present study explores the utility of the ornithine effect to enhance the characterization of cyclic and stapled peptides. By exploiting the predictability of the ornithine effect, unique ions are generated that allow the differentiation of isomers. Both collision-based and photon-based activation methods are used to characterize the modified and unmodified peptides. The fragment ions were assigned using an in-house algorithm designed to systematically generate a list of every possible combination of bond cleavages (including cross-ring cleavages) and the associated masses of the fragments.
Materials and Reagents
HPLC grade water and methanol used for sample preparation and dilution were purchased from EMD Millipore (Billerica, MA, USA) and formic acid was purchased from Fisher Scientific (Fairlawn, NJ, USA). Sunflower trypsin inhibitor-1 (SFTI-1) was provided by Bristol-Myers Squibb (Princeton, NJ, USA). Dithiothreitol (DTT) and iodoacetamide (IAM) used for reduction and alkylation of disulfide bonds were purchased from Sigma Aldrich (St. Louis, MO, USA). Proteomics-grade trypsin was obtained from Promega (Madison, WI, USA). The stapled peptides were synthesized as described in the Supplemental Information.
Synthesis and Purification of Stapled Peptides
Stapled peptides were produced by BioSynthesis (Lewisville, TX, USA) using Fmoc solid phase peptide synthesis. Peptides were stapled by ring-closing metathesis using Grubbs’ First Generation Catalyst, and purified by preparative C18 reversed-phase HPLC. The purity of the peptides was confirmed as >95% by analytical HPLC and by MALDI-TOF MS.
Unmodified samples were diluted to 5 uM with an equal mixture of methanol and water with 1% formic acid prior to direct infusion ESI. Stapled peptides and SFTI-1 were mixed with excess hydrazine hydrate for 4 h at 55 °C in water to promote the conversion of arginine to ornithine. The reaction mixture was dried under vacuum (Thermo Savant DNA 120 Speedvac Concentrator, San Jose, CA, USA) to remove organics and diluted to 5 uM with an equal mixture of methanol and water with 1% formic acid prior to direct infusion ESI. After the incubation of SFTI-1 with hydrazine hydrate, the intramolecular disulfide bond was reduced with dithiothreitol (100 mM in water) for 45 min at 55 °C and alkylated with iodoacetamide (100 mM in water) for 45 min protected from light at room temperature.
Mass Spectrometry and Photodissociation
All MS experiments were performed on a Thermo Scientific Orbitrap Elite mass spectrometer (San Jose, CA, USA) custom fit with an unfocused, non-collimated Coherent ExciStar 193 nm excimer laser (Santa Clara, CA, USA) to perform ultraviolet photodissociation, as previously described [46, 47]. Peptides, both modified and unmodified, were analyzed by direct infusion electrospray ionization with a spray voltage of 4 kV and a capillary temperature of 275 °C. For all CID experiments, the normalized collision energy (NCE) was varied to reduce the precursor to approximately 10%–15% relative abundance during an activation period of 10 ms (this is sufficient to provide rich fragmentation without excessive ejection of the precursor ion during resonance exCitation). For all UVPD experiments, a laser power of 1.5–2.5 mJ/pulse and one to three 5-ns pulses were used. The resulting MS/MS spectra were interpreted manually as well as by using a custom fragment ion prediction algorithm that was developed in-house.
Development of a Custom Algorithm for Assignment of Fragment Ions
Several algorithms have recently been developed to assist with the characterization of cyclic peptides. Two of the most recent algorithms are CYCLONE and CycloBranch, which were developed to characterize cyclic peptides via a de novo strategy [48, 49]. An in-house algorithm was developed in C# to assign fragment ions of cyclic peptides based on interpretation of 193 nm UVPD mass spectra. Unlike CYCLONE and CycloBranch, this algorithm utilizes a naïve approach that calculates all potential fragment ions which may arise from the cleavage of any bond in the peptide’s structure and cross-ring cleavages based on candidate structures. Moreover, the custom algorithm accepts any type of candidate structure, including stapled peptides containing unnatural amino acids, unlike other available algorithms. The structures of the candidate peptides were generated in ChemDraw Perkin Elmer (Waltham, MA, USA). The structures were assigned atom numbers and implicit hydrogens were selected to be hidden. The final structure was saved as a cdxml file.
The XML from the cdxml files were then parsed by the algorithm and converted into an undirected graph (abstract data structure). A naïve approach was employed in which fragments were calculated through the systematic removal of each edge in the graph from the candidate molecule’s structure. Additionally, with a ring size specified, fragments resulting from cross ring cleavages could also be calculated (resulting from the cleavage of two bonds). These calculated fragment ions were saved in a SQLite database , which could be exported into an Excel file. The ions in the MS/MS spectra were then manually compared with the theoretically calculated masses.
Results and Discussion
When analyzing the MS/MS spectra of stapled and cyclic peptides, there is an inherent challenge in nomenclature associated with the fragment ions due to the presence of multiple branches or the lack of any terminal positions. In this study, an in-house algorithm was developed to assist in the identification and assignment of the resulting fragment ions. For the stapled and cyclic peptides, a fragment ion involving the cyclic portion can only be produced via cleavages of two bonds (one in the cyclic region and one in the linear segment, typically creating an internal ion) or via a cross-ring cleavage. A single bond cleavage between atom numbers 1 and 2 is annotated as “[atom number 1 | atom number 2]” and the cleavage of two bonds between atom numbers 1 and 2 and atom numbers 3 and 4 simultaneously is annotated as “[atom number 1 | atom number 2 || atom number 3 | atom number 4]”. If multiple cleavages lead to the same m/z ratio, all possible fragments are listed.
As an alternative to the ornithinylation approach, tryptic proteolysis of SFTI-1 was undertaken. Tryptic digestion of SFTI-1, either with reduction/alkylation or without, failed to cause ring opening. Because SFTI-1 is a known trypsin inhibitor [51, 52], this result was not surprising and emphasizes the need for alternative approaches to facilitate the characterization of unusual peptides.
Conversion of arginine residues to ornithine residues provided an effective way to promote ring opening reactions of stapled or cyclic peptides chemically rather than enzymatically, with greater specificity than tryptic digestion. The preferential heterolytic cleavage C-terminal to the ornithine residue upon activation led to an N-terminal ion that terminated with a six-membered lactam ring. The ornithine effect allowed characterization of isomeric stapled peptides as well as the cyclic peptide SFTI-1 upon CID or UVPD. A custom algorithm was developed to facilitate assignment of mass values to all possible fragment ions created upon cleavage of two bonds, including cross-ring cleavages. Use of this algorithm simplified the interpretation and assignment of fragment ions produced from cyclic peptides.
The authors acknowledge support for this work by the NSF (CHE 1402753) and the Welch Foundation (F-1155).
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