UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A method to characterize formaldehyde cross-linking in proteins by mass spectrometry Ding, Xuan


The formaldehyde cross-linking approach has been used to identify protein interactions in living cells and organisms, and has the potential to map the geometry of interactions based on cross-linked peptides. However, the identification of cross-linked peptides has not been realized in native proteins, not even in model proteins. In this study, a method to identify and characterize cross-linked peptides in model proteins is developed. The method was initially developed in an insulin model system. Candidates of cross-linked peptides were identified by matching a list of putative cross-linked peptides to experimental MS signals. Signals in the MS/MS spectrum of a candidate were matched with proposed fragment ions, and confirmation of all proposed structural components verified a candidate to be a cross-linked peptide. As a result, three cross-linked insulin peptides were identified for the first time. The CID fragmentation of a formaldehyde cross-linked peptide proved to occur at both the cross-link bridge and peptide backbones. Fragment ions containing the cross-link bridge allowed the localization of cross-link sites, which revealed a specific N-terminus to tyrosine cross-link. The method was then refined using two model protein systems of equivalent and higher complexity. Five cross-linked insulin peptides and three cross-linked myoglobin peptides were identified, with cross-link sites localized. The fragmentation patterns of cross-linked peptides were further confirmed. The localization of cross-link sites in proteins revealed the N-terminus to tyrosine/asparigine and lysine to tyrosine cross-links, cross-links on arginine, and two cross-links forming on one single N-terminus. Furthermore, monitoring progression of the two reaction steps at cross-link sites revealed the chemistry of formaldehyde cross-linking reaction in proteins for the first time. In addition, with more complex data as the iii size of the model protein increased, the method was refined by applying programming to data processing and a bar-graph visualization to localize cross-link sites on isomeric peptides. In the future, this method can be applied to other model protein systems for a more comprehensive understanding of formaldehyde cross-linking. The fragmentation patterns and reaction chemistry revealed by this method can be used to facilitate the identification of cross-linked peptides in native proteins.

Item Media

Item Citations and Data


Attribution-NonCommercial-NoDerivs 3.0 Unported