| Summary: | Protein identification generally involves enzymatic digestion of proteins followed by mass spectrometry (MS) sequencing analysis of the resulting peptides, a process referred to as bottom-up proteomics. Another approach (top-down proteomics) consists of MS analysis of intact proteins without prior enzymatic digestion, using Fourier Transform Ion Cyclotron (FT-ICR) mass spectrometry. The latter has the advantage of allowing for 100% sequence coverage, and greater access to post translational modifications. However, these types of analyses are limited by the cost of the MS technology. In addition, FT-ICR fragmentation efficiency decreases as the protein mass increases, hindering direct fragmentation of large proteins. Nevertheless, techniques that facilitate protein fragmentation in the gas-phase have the potential to improve top-down strategies. Our research focuses on promoting site-specific peptide and protein fragmentation in the gas-phase. With this goal, we employed the use of chemical modification of lysine residues with a labile peroxycarbamate group, which upon collision induced dissociation (CID) decomposes to initiate a radical mediated fragmentation pathway at or near the modified lysine. This peroxycarbamate chemistry was extended to the modification of peptides N-termini. N-terminal peroxycarbamates lead to cleavage of the N-terminal amino acid side chain upon CID, thus identifying this residue. This fragmentation also lead to the labeling of the b-ion series by a mass shift equal to the mass of the N-terminal side chain when compared to unmodified peptides. These results offer advantages in de novo sequencing analyses as well as peptide mass fingerprinting methods.
The application of the hydrazinolysis of proteins as a chemical protein digestion technique was also investigated. These studies lead to the development of hydrazinolysis conditions that allow for partial degradation of proteins to produce large fragments that are within the ideal mass rage for direct fragmentation with FT-ICR MS. This chemical digestion has the advantage over enzymatic digestion in that it is possible to retain 100% sequence coverage. All of these findings open a new door for the improvement of top-down proteomics.
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