Introduction
Cyclic peptides represent a middle ground between small and large-molecule drugs, and often possess the easier synthesis and delivery of small molecules yet the specificity of large molecules. Cyclic peptides pose some analytical challenges including non-ribosomal peptides (not in the genome), non-standard amino acids, both L and D-amino acids, numerous post-translational modifications (PTMs), and disulfide and other bridges.
Here we describe how to use Byonic to sequence cyclic peptides.
Methods
Create FASTA file
The difficulty in characterizing cyclic peptides is the lack of inherent N/C-terminals and the diverse types of cyclization. How to define the start and end residues of cyclic peptides in the fasta file is the key to a successful search. Byonic's strategy is to linearize cyclic peptides based on the direction of the peptide bond (i.e. the direction of the arrow in the structural diagram).
Generally, cyclic peptides contain non-standard amino acids. Byonic/Byos can support non-natural amino acid sequence analysis. You can obtain the more information on the workflow settings containing non-natural amino acids here.
① Disulfide bond cyclization
Somatostatin is a cyclic peptide connected by a disulfide bond (i.e., the curve in the structural diagram). Byonic defines the disulfide bond as a modification, whereby Somatostatin can produce only one linear peptide. The fasta file is shown in Figure 2:
Figure 1 Structural diagram of Somatostatin
Figure 2 Linear sequence of Somatostatin
② Head-to-tail cyclization
Unguisin A is a cyclic heptapeptide, which can be randomly opened at each amide bond to produce 7 linear peptides. GABA is a non-standard amino acid, and Byonic can add a fixed modification to replace GABA with residue J (precise molecular weight is 100). The fasta file is shown in Figure 4:
Figure 3 Structural diagram of Unguisin A
Figure 4 Linear sequence of Unguisin A
③ Side chain amino group to the C-terminal carboxyl group cyclization
Polymyxin B1 contains a large amount of non-standard amino acid Dab (2,4-diaminobutyric acid) and a fatty acid. Byonic replaces the Dab residue and fatty acid residue with X and J respectively. The fasta file is shown in Figure 6:
Figure 5 Structural diagram of Polymyxin B1
Figure 6 Linear sequence of Polymyxin B1
④ Side chain carboxyl group to the N-terminal amino group cyclization
Siamycin II is a linear side chain cyclic peptide containing two disulfide bonds. According to the direction of the peptide bond, one linear peptide can be generated. The fasta file is shown in Figure 8:
Figure 7 Structural diagram of Siamycin II
Figure 8 Linear sequence of Siamycin II
Parameters setting
- Protein database options
The Add decoys box should be unchecked, as some true-positive PSMs will be filtered out because of the high complexity of cyclic peptides:
- Sample digestion
Leave Cleavage site(s) as empty for Top-Down searches:
- Modifications
① Disulfide bond cyclization
Under the S-S, Xlink option, check Disulfides. If the amino acids are coupled through a cross-link reagent, check Crosslink: Custom, then enter the delta mass and cross-link sites according to the format:
② Head-to-tail cyclization
Unguisin A contains a non-standard amino acid GABA, which needs to be set as a fixed modification. A variable modification of dehydration is set at the C-terminus of the cyclic peptide because the cyclic peptide may be linearized in vitro. This setting allows Byos to search for all possible linearized peptides:
③ Side chain amino group to the C-terminal carboxyl group cyclization
Polymyxin B1 contains two non-standard amino acids, FA and Dab, which can be replaced by J and X respectively, and set as fixed modifications. Enable wildcard search to find all possible unknown modifications:
④ Side chain carboxyl group to the N-terminal amino group cyclization
Byonic treats disulfide bonds as rare modification default. Siamycin II contains two disulfide bonds, so the total rare max needs to be changed to 2:
Result view
Byos automatically selects the linear peptide that best matches the spectrum, which usually has the highest score. In addition to b/y ions, Byos also automatically identifies internal fragments, such as the peptide AGCKNFFWKTFTSC, i(5-12) indicates the internal fragment NFFWKTFT. Note: In the current commercial version, internal fragments are not included in the scoring mechanism:
Summary
Characterization of cyclic peptides by MS2 is significantly more challenging than elucidation of linear peptides. Cyclic peptides require cleavage of two backbone bonds to generate fragment ions, and the absence of intrinsic N-terminal and C-terminal positions complicates spectral interpretation. While several data acquisition options including MSn methods and / or alternative activation methods like UVPD have demonstrated ability to analyze these peptides, the bottleneck of the workflow has been the spectral interpretation, requiring laborious manual analysis.
We have demonstrated that Byonic/Byos can search, identify and annotate cyclic peptides. We foresee the advantages of incorporating cyclic peptide search into proteome search engines, such as distinguishing MS-cleavage cyclic peptides from in vitro cleavage cyclic peptides, and searching for sequence variants, unknown modifications, and non-standard amino acids through wildcard search.