Radical-Driven Tandem Mass Spectrometry: Improved Analytical Strategies and Mechanistic Studies for Characterization of Labile Biomolecules

Posttranslational modifications (PTMs) have significant impacts on structure and function of proteins. Thus, characterization of PTMs is essential for understanding their roles in disease progression. Mass spectrometry is a powerful tool for PTM analysis. This research involves the development of novel approaches for structural characterization of labile biomolecules, including labile PTMs. Through innovation in radical-driven tandem mass spectrometry (MS/MS), analytical obstacles are resolved and ultimately allow the answering of long-standing questions surrounding protein modifications. Throughout this thesis, a variety of PTM-containing peptides with different chemical and physical properties, ranging from hydrophilic glycopeptides to hydrophobic palmitoylated peptides, are examined, including evaluation of appropriate separation techniques for liquid chromatography mass spectrometry (LC/MS) analysis. Specifically, Chapter 2 discusses how post-column “supercharging” in nanoflow LC/MS improves glycopeptide analysis in several MS/MS techniques, including high energy collision dissociation (HCD), electron transfer dissociation (ETD), HCD product dependent ETD (HCDpdETD), and ETD followed by HCD (EThcD). Supercharging enhanced the average charge state of N-glycosylated peptides from 3.00 to 3.39 and resulted in a significant increase in fragmentation from 6.25% to 93.75%. Chapter 3 extends this work to lipid-containing peptides, including optimization of nanoflow C8 chromatography for such hydrophobic analytes. We also considered protocols for preparation of lipidated peptides with minimal sample loss for direct detection of this PTM. For example, with supercharging, the N-Ras C-terminal peptide was directly detected due to an enhanced charge state from 1.59 to 2.31. This charge enhancement allowed ETD analysis of the triply protonated peptide, resulting in extensive fragmentation corresponding to ~95% sequence coverage without loss of farnesyl or the C-terminal O-methyl group. Chapter 4 discusses the extension of collision induced unfolding (CIU) coupled with ion mobility spectrometry towards PTM-carrying peptides. This approach allows observation of gas-phase structural transitions and direct analysis of how such transitions affect electron capture dissociation (ECD) sequence coverage. Intriguingly, unfolding of such species proceed via minimum PTM loss and rich ECD fragmentation patterns (i.e., ~82% sequence coverage) are observed for the resulting extended conformations. Replica exchange molecular dynamics was performed to better understand this unfolding process for phosphorylated peptides. In Chapter 5, the CIU-ECD approach from Chapter 4 is extended towards monoclonal antibodies (mAbs) for middle-down analysis of their glycosylation. With CIU-ECD, sequence coverage for Fc dimer fragments were greatly improved from 14% to ≥30%. Through this work, we discovered that supercharging could stabilize protein structure prior to CIU for improved CIU fingerprints. An automated 2D-LC online digestion setup was developed for middle-down digestion of mAbs. Such automation improves digestion reproducibility and reduces human error. Finally, Chapter 6 discusses the extension of negative ion ECD (niECD) to a new class of molecules, gangliosides. Such analytes are acidic and benefit from negative ion mode analysis. We found that niECD outperforms both collision induced dissociation (CID) and electron induced dissociation (EID) for GM1, GM3, GD1a and GD1b. Furthermore, through unique radical ion chemistry, niECD allowed differentiation of isomeric gangliosides. Overall, the research presented in this dissertation improves our understanding of radical-driven tandem mass spectrometry for improved analysis of labile biomolecules. This work enables new analytical tools for structural characterization of biological samples.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/172587/1/nhle_1.pd