High Resolution Peptide Tandem Mass Spectrometry: Alternative Charge Carriers in Electron-based Activation Methods and Improved Antibody Sequence Variant Analysis.

Abstract

Electron-based tandem mass spectrometry (MS/MS) techniques, e.g., electron capture dissociation (ECD), electron transfer dissociation and electron detachment dissociation (EDD) have shown unique advantages in biomolecular analysis, e.g., for post-translational modification site determination. The mechanisms of such EXD techniques are debated; however, location and chemical environment of proton charge carriers are agreed to be important. Alternative charge carriers, e.g., metal ions and salt anions, are often present; however only limited information on their EXD influence is available. , We systematically investigated how metal-peptide interactions affect ECD outcome in positive ion mode. We found that stronger metal-peptide interaction results in higher ECD fragmentation efficiency, presumably due to metal shielding from the incoming electron. However, for multiple metal attachments to one peptide, metal ions appear to bind complementary ECD fragment pairs together, thus preventing them from being released and observed. We also investigated the possibility of salt anion adduction improving EDD performance in negative ion mode. Bromide adduction appears promising although a similar issue is apparent in that product ion pairs do not appear to separate and thus are not observed. Additional vibrational activation will be necessary to further explore this approach. We also expanded MS/MS analysis to a pharmaceutical research context. A targeted liquid chromatography (LC)/MS/MS workflow was developed for analysis of pharmaceutical protein sequence variants. With this improved workflow, we achieved higher sensitivity in sequence variant detection, fast false positive screening, and DNA error determination. Information provided by this workflow can be used for characterizing pharmaceutical protein products, as well as for optimizing protein production. In an effort to facilitate these research projects, and to ensure reproducible data analysis, three data analysis programs were developed. The purposes of these programs include: MS/MS spectral analysis for alternative charge carriers, LC/MS searching for low-abundance peptide ions, and false positive removal of LC/MS/MS protein sequence variant search results using wild type decoys. Overall, research presented in this thesis contributes to improved understanding of electron-based MS/MS methods with alternative charge carriers, and to enhanced use of MS/MS in pharmaceutical research and development.