Studies of protein post-translational modifications using high resolution tandem mass spectrometry

Abstract

Electron capture dissociation (ECD) is a powerful and superior tandem mass spectrometry (MS) fragmentation technique in the study of protein post-translational modifications (PTMs) due to its unique features of preserving labile modifications and providing more detailed sequence information, which has been used to study protein platination and disulfide linked proteins. Cisplatin was found cross-linking multiple methionine (Met) pairs on calmodulin (CaM). The cross–linking of cisplatin to apo–CaM or Ca–CaM can inhibit the ability of CaM to recognize its target proteins as proved by a melittin binding assay. To further establish MS strategies to quickly assign the platinum-modification sites, a series of peptides with potential cisplatin binding sites were reacted with cisplatin and then analyzed by ECD. Radical-mediated side chain losses from the charge-reduced M+Pt species (such as CH3S• or CH3SH from Met, SH• from Cys, CO2 from Glu or Asp, and NH2• from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-modification sites on certain amino acid residues. Furthermore, the potential of cisplatin as a protein crosslinking reagent was further explored and demonstrated on other peptides and proteins. Many of the inherent features of cisplatin make it an interesting cross-linking reagent, such as targeting new protein functional groups (thioether and imidazole groups), its unique isotopic pattern, its inherent positive charges, its potential of binding to different functional groups, etc. However, it was found that the distance constraints obtained from NMR structures of CaM are inconsistent with the measured distance constraints by cross–linking. Therefore, a newly developed flexibility simulation method was applied to explore whether the flexibility motions of CaM might contribute to the observed Pt-crosslinking on CaM. The flexibility analysis showed that the structural flexibility of CaM is key to cisplatin crosslinking CaM. ECD mechanism of disulfide bonds is still under debate. To further explore the ECD mechanism of sulfur– containing species, a series of disulfide (S–S), sulfur–selenium (S–Se), and diselenide (Se–Se) bond–containing peptides was studied by ECD. The results demonstrate that the radical has higher tendency to stay at selenium rather than sulfur after cleavage of Se–S bonds by ECD and suggest that direct electron capture at Se–Se and C–Se bonds is the main process during ECD of inter–chain diselenide peptides. Last but not least, a new active ion ECD (AI-ECD) method, named Shots-ECD, was developed and applied to improve Top-down ECD backbone fragmentation efficiency of disulfide-rich proteins. The results show that the Shots–ECD approach can not only cleave multiple disulfide bonds but also significantly improve the backbone cleavage efficiency. This strategy is fast, efficient, and with no need of chemical reduction of samples and instrument modification, and therefore can be a powerful approach to improve top-down ECD efficiency of not only disulfide bonded proteins but all proteins by Fourier transform ion cyclotron mass spectrometry (FTICR MS)