Measuring a single MS2 spectrum is the simplest way to identify the peptides in a crosslink. However, it results in a large number of fragments that might belong to four or more different peptide sequences. The correct interpretation of these results is challenging and might decrease the confidence of the identification. The number of sequences depends on the crosslinker and fragmentation technique used:

  • MS non-cleavable crosslinker (or crosslinker not cleavable by the used fragmentation technique)—The spectrum contains two peptide fragment series:
    • One for peptide A with the crosslinked peptide B treated as a modification
    • One for peptide B with the crosslinked peptide A treated as a modification
    • The spectrum display highlights both fragment series. Depending on the fragmentation of the crosslinked peptides, the construct might show several internal fragments with two broken peptide bonds. The application does not highlight these internal fragments.
  • MS-cleavable crosslinker—When the application uses a cleavable crosslinker with two internal cleavage sites, the view displays five different peptide ion series. Each peptide comes with two different modifications for the broken crosslinker, depending on the random cleavage on one of the two cleavage sites in the crosslinker. In addition, the application displays unfragmented peptides after cleavage of the crosslinker as extra fragment series.

The color coding of single peptides after the crosslinker is broken and the fragment ions are used is as follows:

  • Peptides—peptide A orange/red, peptide B blue
  • Fragment series of peptide A—orange and red
  • Fragment series of peptide B—medium blue and light blue

The following figure shows an example of a cleavable crosslinker spectrum in a single MS2 fragmentation spectrum. The Crosslink Spectrum Match Identification Details dialog box shows fragment series of peptides A and B and reporter ions.

Cleavable crosslinker spectrum in a single M2 fragmentation spectrum

The following figure shows a fragment found twice in the spectrum. The crosslinker might break at different positions, resulting in two different peptide fragments with the same sequence but with different modifications at the crosslink sites. Therefore, the fragment spectrum can show different peaks for the same fragment (b5). The two fragments show a mass difference of 31.97 Da, which corresponds to the difference between the alkene and the thiol fragment of the crosslinker.

Spectrum showing different peaks for the same fragment