MS Cleavable Crosslinkers and Tags for Protein Structural Analysis.

Abstract

Chemical crosslinking combined with mass spectrometry is a powerful tool for analyzing protein interactions, however, there are several challenges associated with the method. Proteins are frequently crosslinked at lysine residues which makes the proteins more difficult to digest, resulting in large, branched peptides that can be difficult to ionize and fragment. Data interpretation is difficult because there are two overlapping series of ions from each peptide in the MS/MS spectrum. The first goal of this thesis was to synthesize, evaluate, and apply new crosslinkers to overcame several of these challenges. The first crosslinker synthesized, PC1, was a cyclic tertiary amine and exhibited several of the desired properties upon initial evaluation with model peptides. However, upon application to the tetrameric protein aldolase, little fragmentation of the peptide backbone was needed for crosslinked peptide identification. The structure of PC1 was then modified to make the crosslinker more labile and the crosslinkers DC4, DC5, and DC6 were synthesized. DC4, DC5, and DC6 each contained two intrinsic positive charges due to the presence of two quaternary amines. DC4-crosslinked peptides were characterized and facile fragmentation was observed at both sites of positive charge. This yielded sets of ions differing by the mass loss from the quaternary amines, and produced a mobile proton needed for backbone fragmentation. Further CID of the fragments consistently provided spectra suitable for peptide identification. DC4 was applied to the protein aldolase and several crosslinked peptides were found that were in agreement with the crystal structures. Residues that were deeply buried within the crystal structure were not observed as modified. DC4-crosslinked proteins were analyzed by ion-mobility mass spectrometry. Crosslinked proteins had a larger CCS and required higher energies to unfold and to dissociate, indicating that crosslinkers stabilize the quaternary and tertiary structures of proteins. Crosslinking with DC4 altered the fragmentation pathway because different subunits were dissociated from the tetramer of crosslinked avidin than unmodified avidin. Furthermore, when sufficient energy was applied to fragment the tetramer into peptides, crosslinked avidin resulted in a much richer series of unique peptides compared to noncrosslinked avidin.