Native Ion Mobility–Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level

Abstract

Protein sialylation has been closely linked to many diseases including Alzheimer’s disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility–mass spectrometry (IM–MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM–MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM–MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM–MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.

Comparing Selected-Ion Collision Induced Unfolding with All Ion Unfolding Methods for Comprehensive Protein Conformational Characterization

“…The charge stripping phenomenon observed during IMS–MS analysis was evaluated using antibodies by Vallejo et al where they noted that charge stripping occurs predictably based on charge state, with lesser charged ions experiencing reduced charge stripping . In addition, our recent application of AIU to sialylated glycoproteins independently support the limited influence of charge stripping on certain systems . Based on these observations, we expect the interference of charge stripped products to be limited to below 5% of the intensity of the precursor ion, which might not be the contributing factors preventing the further application of CCS acc and AIU strategy, although this basically requires many more practices and validations adding to the proof-of-concept demonstration of the current study.…”

“…44 In addition, our recent application of AIU to sialylated glycoproteins independently support the limited influence of charge stripping on certain systems. 53 Based on these observations, we expect the interference of charge stripped products to be limited to below 5% of the intensity of the precursor ion, which might not be the contributing factors preventing the further application of CCS acc and AIU strategy, although this basically requires many more practices and validations adding to the proof-of-concept demonstration of the current study. To this end, we recommend performing a pretest for potential charge stripping effects would be a practical solution for broader application of AIU and CCS acc algorithm.…”

Expedited Evaluation of Conformational Stability–Heterogeneity Associations for Crude Polyclonal Antibodies in Response to Conjugate Vaccines

“…Following the comprehensive characterization of molecular heterogeneity, we then evaluated the conformational stability of cpAbs. Gas-phase unfolding technique, namely, CIU, has previously been well suited to gather the conformational and dynamic structural information on isolated IgG in a charge state-dependent data acquisition mode. ,− To further empower the unfolding technique in terms of data efficiency and signal-to-noise ratios, we employed an updated data acquisition and visualization package for CIU, recently published as the AIU method. , In addition to the first application of AIU to the crude antibody for this study, we inherit the merits of CIU and CIUSuite software tools developed by the Ruotolo group , and proposed herein for the first time the use of unfolding fingerprints averaging from multiple biological replicates from different mice to perform quantitative comparisons (Figure S4), through the generated AIU50 values and RMSD values across different batches of cpAb samples. While averaged feature CCS values can deliver overall structural information for each identified unfolding intermediate, the AIU50 and RMSD values permit the direct readout of conformational stability and heterogeneity, respectively …”

“…In this work, we combined two cutting-edge MS-based approaches, i.e., (1) expedited, quantitative CIU-IM-MS technique with all-ion mode, namely, All Ion Unfolding (AIU), , and (2) bottom-up de novo sequencing and glycoproteomic profiling MS technique, for the conformational and compositional quantification of the heterogeneity and stability alteration of four groups of comparative crude polyclonal antibody (cpAb). Figure S1 illustrates the overall workflow for the comprehensive characterization of the carrier protein-dependent heterogeneity-stability relationship of cpAbs in response to four different vaccine conditions.…”

“…Meanwhile, ion mobility-mass spectrometry (IM-MS) is capable of separating gas-phase protein ions according to their size, shape, and charge (frequently converted to orientationally averaged collision cross section (CCS)) and can thus act as a tool to resolve ions that may be indistinguishable by mass alone or to achieve indispensable structural information at the intact protein level, − making it suitable for global heterogeneity characterization of intact antibodies. Collision-induced unfolding (CIU) has effectively enriched the IM-MS toolbox through potentially improved resolution in CCS and associated unfolding intermediate information for rapid protein structural and conformational analysis in the gas phase. − Many prior studies have successfully proven the power of the CIU-IM-MS regime in rapid quality assessment of monoclonal antibodies, mainly focusing on conformational stability and CCS variations in response to either extra physical stress or chemical treatments. − Hybrid experiments have identified key structural elements of IgG monoclonal antibodies, including glycosylation, disulfide bridge, domain exchange, and even batch-to-batch variations. − This work expands on those applications by assessing the structural heterogeneity of polyclonal antibody responses across biological replicates and the driving force behind this type of heterogeneity.…”