Summary:
For a crosslinked peptide pair assignment, this article explains:
- How Byos chooses the Sequence and Xlink Partner Peptide Sequence designations for each of the crosslinked peptides.
- And which peptide the Score and Xlink Score correspond to.
Introduction:
Byos supports the detection and assignment of crosslinked peptide pairs, allowing MS/MS assessment of the two crosslinked peptides at once. Byos reports both peptide sequences and MS/MS Byonic scores with the fields Sequence, Xlink Partner Peptide Sequence, Score, and Xlink Score. Byos must decide which of the peptide crosslink peptide pairs gets assigned to each field. The logic dictating how these fields are populated is designed to simplify assessment and reporting of results.
NOTE: Byos can detect >2 crosslinked peptides, but only assesses MS/MS for two of the peptide sequences. Any additional crosslinked peptides are detected as MS1 parent ion mass shifts without MS/MS analysis.
Sequence vs. Partner Peptide Sequence:
When a crosslinked peptide pair is identified, one peptide sequence is reported with the field Sequence while the other peptide sequence is reported with the field Xlink Partner Peptide Sequence. The peptide sequence that is earliest in the workflow protein sequence list is assigned as Sequence. The disulfide bridged peptide pair assignment in Figure 1 is used as an example to help explain how this logic works.
Figure 1: Example crosslinked peptide pair identified in Byos S-S.
If the peptide pair in Figure 1 represents an intra chain disulfide bond, the VYACEVTHQGLSSPVTK peptide sequence should be closer to the N-terminus than the LSCVASGFIFSNHWMNWVR peptide sequence, as shown in the Figure 2 example protein sequence.
Figure 2: Example protein sequence to help explain Byos crosslinking assignment logic for an intra chain disulfide bond. The peptide sequence that is closest to the N-terminus is reported as Sequence, while the peptide sequence closest to the C-terminus is reported as Xlink Partner Peptide Sequence.
If the peptide pair in Figure 1 represents an inter chain disulfide bond, the VYACEVTHQGLSSPVTK peptide sequence should be assigned to a protein sequence appearing earlier the Byos workflow Proteins table, as shown in Figure 3.
Figure 3: Example workflow to help explain Byos crosslinking assignment logic for an inter chain disulfide bond. The peptide sequence assigned to the protein appearing first in the workflow Proteins table is reported as Sequence, while the peptide sequence assigned to the protein appearing later in the workflow Proteins table is reported as Xlink Partner Peptide Sequence.
An exception to this rule presents when a variable modification is detected on the peptide sequence that would typically be assigned as the Xlink Partner Peptide Sequence. This is because Byos does not allow the Xlink Partner Peptide Sequence to have a variable modification. For example if Byos detects VYACEVTHQGLSSPVTK disulfide bonded to LSCVASGFIFSNHWmNWVR with oxidation identified as a variable modification on methionine, LSCVASGFIFSNHWMNWVR would be assigned as Sequence regardless of its location in the workflow protein sequence list relative to VYACEVTHQGLSSPVTK.
Score vs. Xlink Score:
When a crosslinked peptide pair is identified, one peptide MS/MS Byonic score is reported as Score while the other peptide MS/MS Byonic score is reported as Xlink Score. The highest MS/MS Byonic score is always reported as Score, while the lowest MS/MS Byonic score is always reported as Xlink Score.