Collision-induced dissociation of lys-lys intramolecular crosslinked peptides |
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Authors: | Amadeu H Iglesias Luiz F A Santos Fabio C Gozzo |
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Institution: | 1. Center for Structural and Molecular Biology, Brazilian Synchrotron Light Source, Campinas, Brazil 2. Institute of Chemistry, State University of Campinas-UNICAMP, CP 6154, 13083-970, Campinas, SP, Brazil
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Abstract: | The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry
to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of
detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked
peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species
found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence
of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion
yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted
laser desorption/ionization-generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation
starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide
bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified
peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation
of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other
hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product
ion containing the crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data
analysis of crosslinked peptides. |
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