Advancing Intact Protein Analysis By Top Down Mass Spectrometry PDF Download

The interrogation of protein structure, especially identifying and localizing post-translational modifications (PTMs) and sites of ligand/small molecule binding, is crucial for understanding protein function in biological systems. In particular, the rapid increase of the use and development of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) for human health have necessitated the advancement of efficient and accurate analytical methods. Top-down and middle-down mass spectrometry (TD- and MD-MS) have become prominent analytical tools for protein characterization. However, obtaining complete protein sequence coverage by TD-/MD-MS can be limiting, particularly for proteins greater than 30 kDa, e.g., mAbs and ADCs. My dissertation research explores the utility of internal fragments, which are largely ignored by the MS community as they are difficult to assign, from both fundamental and application perspectives, for more efficient and comprehensive protein sequence, structure, and PTM characterization. On the fundamental side, we demonstrated a relationship between internal fragments and conventional terminal fragments. A better understanding of the fundamental formation mechanism of internal fragments aids the development of sequencing algorithms to assign internal fragments more accurately and reliably. On the application side, we started by analyzing standard disulfide-intact proteins using TD-MS, in which assigning internal fragments helped achieve near complete sequence coverage, and obtain disulfide bond position and connectivity information. This encouraged us to apply TD-MS on proteins of therapeutic significance, i.e., mAbs and ADCs, which are extremely complex disulfide-intact proteins. Incorporating internal fragments analysis allowed us to achieve the highest sequence coverage to date on an intact mAb by TD-MS, and enabled the access of important PTM information and drug binding information on intact ADCs. We then expanded this application by applying MD-MS on reduced mAbs and ADCs. Analyzing internal fragments in MD-MS helped us achieve comprehensive characterization of mAbs and ADCs which is a significant improvement from TD-MS and is comparable to the results obtained from the routinely utilized bottom-up peptide mapping method. Lastly, we demonstrated that assigning internal fragments generated by collision-based fragmentation also helps deliver higher-order structure information of multi-subunit protein assemblies. In summary, this dissertation work contributes to advancing the technique and instrumentation surrounding TD- and MD-MS workflows to achieve better characterization of proteins, especially biotherapeutics, and identification of specific proteoforms.