Internal Fragments in Top-down and Middle-down Mass Spectrometry: From Fundamentals to Applications

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

Top-Down Mass Spectrometry and Assigning Internal Fragments for Determining Disulfide Bond Positions in Proteins

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS) where only terminal fragments are assigned, for disulfide intact proteins can access disulfide information, but suffers from low fragmentation efficiency, limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into TD-MS workflow for analyzing disulfide intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry

Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complimentary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum.</p

Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins.

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates

Towards understanding the formation of internal fragments generated by collisionally activated dissociation for top-down mass spectrometry

Top-down mass spectrometry (TD-MS) generates fragment ions that returns information on the polypeptide amino acid sequence. In addition to terminal fragments, internal fragments that result from multiple cleavage events can also be formed. Traditionally, internal fragments are largely ignored due to a lack of available software to reliably assign them, mainly caused by a poor understanding of their formation mechanism. To accurately assign internal fragments, their formation process needs to be better understood. Here, we applied a statistical method to compare fragmentation patterns of internal and terminal fragments of peptides and proteins generated by collisionally activated dissociation (CAD). Internal fragments share similar fragmentation propensities with terminal fragments (e.g., enhanced cleavages N-terminal to proline and C-terminal to acidic residues), suggesting that their formation follows conventional CAD pathways. Internal fragments should be generated by subsequent cleavages of terminal fragments and their formation can be explained by the well-known mobile proton model. In addition, internal fragments can be coupled with terminal fragments to form complementary product ions that span the entire protein sequence. These enhance our understanding of internal fragment formation and can help improve sequencing algorithms to accurately assign internal fragments, which will ultimately lead to more efficient and comprehensive TD-MS analysis of proteins and proteoforms

Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody-Drug Conjugates.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789