Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets

Electrospray ionization coupled to mass spectrometry is a key technology for determining the stoichiometries of multiprotein complexes. Despite highly accurate results for many assemblies, challenging samples can generate signals for artifact protein–protein binding born of the crowding forces present within drying electrospray droplets. Here, for the first time, we study the formation of preferred protein quaternary structures within such rapidly evaporating nanodroplets. We use ion mobility and tandem mass spectrometry to investigate glutamate dehydrogenase dodecamers and serum amyloid P decamers as a function of protein concentration, along with control experiments using carefully chosen protein analogues, to both establish the formation of operative mechanisms and assign the bimodal conformer populations observed. Further, we identify an unprecedented symmetric collision-induced dissociation pathway that we link directly to the quaternary structures of the precursor ions selected

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase–zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody–drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes

Selective Reduction of Cysteine Mutant Antibodies for Site-Specific Antibody–Drug Conjugates

Developing site-specific conjugation technologies for antibody–drug conjugates (ADCs) aims to produce more homogeneous and controlled drug-loaded ADCs to reduce variability and thereby improve the therapeutic index. This article presents a technology that uses cysteine mutant antibodies and mild phosphine-based reductants to prepare site-specific ADCs. The two types of cysteine mutant antibodies, designated C6v1 and C6v2, have one of the interchain disulfide-forming cysteines in the Fab region in the light chain (LC214) or in the heavy chain (HC220) substituted by alanine (or other amino acids), respectively. Certain phosphine-based reductants were found to selectively reduce the “unpaired” cysteines, at the heavy chain (HC220) for C6v1 or at the light chain (LC214) for C6v2 while keeping the interchain disulfide bonds in the hinge region intact, resulting in 90% of DAR2 species and more than 95% of the desired specific conjugation at HC or LC following conjugation to maleimide moieties. The reduction method shows consistent selectivity toward various C6v1 or C6v2 antibody backbones. Sensitivity toward buffer pH for some reductants can be used to optimize reductant reactivity and selectivity. The technology can be further expanded to generate site-specific DAR4 or dual-payload ADCs based on C6v1 or C6v2 antibodies. This technology offers a method to control drug-loading and conjugation sites using a mild one-pot process, as compared to the reduction–oxidation methods used in technologies such as THIOMAB, and shows superior DAR profiles and process simplification as compared to other selective reduction methods

Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures

Monoclonal antibodies (mAbs) are among the fastest growing class of therapeutics due to their high specificity and low incidence of side effects. Unlike most drugs, mAbs are complex macromolecules (∼150 kDa), leading to a host of quality control and characterization challenges inherent in their development. Recently, we introduced a new approach for the analysis of the intact proteins based on ion mobility-mass spectrometry (IM-MS). Our protocol involves the collision induced unfolding (CIU) of intact antibodies, where collisional heating in the gas-phase is used to generate unfolded antibody forms, which are subsequently separated by IM and then analyzed by MS. Collisional energy is added to the antibody ions in a stepwise fashion, and “fingerprint plots” are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. In this report, we have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonding and general levels of glycosylation. In addition, we validate our CIU protocols through control experiments and systematic statistical evaluations of CIU reproducibility. We conclude by projecting the impact of our approach for antibody-related drug discovery and development applications

Bound Anions Differentially Stabilize Multiprotein Complexes in the Absence of Bulk Solvent

The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics, and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility–mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting ‘shell’ of counterions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility–mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of 12 different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counterions on gas-phase protein structure and allows us to rank and classify counterions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride, and nitrate anions are among the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion–protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counterion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent