More than Mass to Charge: Innovative Applications of Mass Spectrometry in Protein Studies
- Author(s): Hamdy, Omar;
- Advisor(s): Julian, Ryan R;
- et al.
Over the past two decades, Mass Spectrometry (MS) has advanced to become an indispensable analytical tool in protein analysis. While the role of proteins in critical biological pathways is well established, their potential as powerful therapeutics has only begun to be realized. This discrepancy motivates the development of novel analytical techniques for protein characterization. The focus of this dissertation is the development of novel MS-based techniques with application in protein studies. The identification of antioxidant peptides/proteins and ionization of proteins under native conditions are the two principle foci of this dissertation.
Maintaining redox homeostasis, or the balance of oxidant and antioxidant forces, is essential for proper cellular functioning in biology. The identification of all contributing antioxidant species is necessary to understand this critically sensitive balance of redox active species. Solution-phase methods used to identify antioxidant peptides require a large amount of sample and are very time-consuming. An MS-based method for the identification of antioxidant peptides is thus desirable and is explored herein. Radical directed dissociation (RDD) is a method developed in the Julian laboratory that utilizes radical chemistry to direct site-specific fragmentation of peptides. RDD is employed to compare the different fragmentation patterns for peptides with known and unknown antioxidant capacities. Antioxidant peptides displayed limited to no evidence of radical directed fragmentation, suggesting that these peptides have a unique ability to sequester and limit migration of the radical. A good correlation between gas phase and solution phase experiments is observed. However, gas-phase experiments are approximately tenfold faster and require significantly less sample.
Extending RDD to the investigation of the antioxidant capacity of peptides from an enzymatic digest of Human Serum Albumin (HSA) revealed four new antioxidants. These findings sparked the work in chapter 3 which sought to identify antioxidant peptides in different proteins with distinct localizations. The presence of antioxidant peptides in all five proteins investigated suggests that the ability to sequester radicals may be common to many proteins as a preservation mechanism to survive exposure to radical containing reactive species (RS). In addition, a detailed analysis of antioxidant peptides reveals interesting findings. Perhaps most surprisingly, is the lack of methionine and cysteine (sulfur containing) residues in many of the antioxidant peptides identified.
The second focus of this dissertation discusses our current understanding of different ionization sources (namely, ESI and liquid-DESI) with an emphasis on achieving “native” conditions for the analysis of proteins via MS. Selective non-covalent adduct protein probing (SNAPP) is utilized to examine protein structural evolution in both liquid DESI and standard ESI under a variety of conditions. Experiments with Cytochrome C (Cytc) demonstrated that methanol induced conformational shifts previously observed with ESI are also readily observed with liquid DESI. The effects of ammonium acetate buffer on liquid DESI SNAPP experiments were examined by monitoring structural changes in myoglobin. Heme retention and SNAPP distributions were both preserved better in liquid DESI than traditional ESI, suggesting superior performance for liquid DESI in buffered conditions. Collectively, the work presented herein contributes to and expands the toolbox of mass spectrometry in protein characterization and has demonstrated considerable potential that merits further exploration utilizing these techniques.