Full Separation and Sequencing of Isomeric Proteoforms in the Middle-Down Mass Range Using Cyclic Ion Mobility and Electron Capture Dissociation.

Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.

Toward Rapid Aspartic Acid Isomer Localization in Therapeutic Peptides Using Cyclic Ion Mobility Mass Spectrometry.

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

High-Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.