Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry

Abstract

Ion mobility-based collision induced unfolding (CIU) has gained increasing attention to probe gas-phase unfolding of proteins and their noncovalent complexes, notably for biotherapeutics. CIU detects subtle conformational changes of proteins and emerges as an attractive alternative to circumvent poor IM resolution. However, CIU still lacks in automation for buffer exchange and data acquisition, precluding its wide adoption. We present here an automated workflow for CIU experiments, from sample preparation to data interpretation using online size exclusion chromatography coupled to native ion mobility mass spectometry (SEC-CIU). Online automated SEC-CIU experiments offer several benefits over nanoESI-CIU, among which i) improved and fast desalting compared to manual buffer exchange used for classical CIU experiments; ii) drastic reduction of the overall data collection time process along with iii) maintaining the number of unfolding transitions. We then evaluate the potential of SEC-CIU to distinguish monoclonal antibodies (mAbs) subclasses, illustrating the efficiency of our method for rapid mAb subclass identification at both intact and middle levels. Finally, we demonstrate that CIU data acquisition time can be further reduced either by setting up a scheduled CIU method relying on diagnostic trap collision voltages or by implementing mAbs-multiplexed SEC-CIU analyses to maximize information content in a single experiment. Altogether, our results confirm the suitability of SEC-CIU to automate CIU experiments, particularly for the fast characterization of next generation mAb-based products.

Pushing the limits of native MS: Online SEC-native MS for structural biology applications

“…Upon addition of SEC dimension, the flowrate is increased significantly (between 100 and 300 µL/min) implying the use of higher desolvation gas and source temperatures to improve solvent evaporation and hence, enhance nMS data. The latter parameter has been recently reported in the literature to have an impact on mAb gas-phase energetics by increasing the internal energy of the ions 33 . These results suggest that source temperatures, and desolvation gas temperatures, need to be carefully adjusted to avoid further source activation and/or fragmentation.…”

“…Van der Rest et al first demonstrated that CCS values of reference proteins obtained using SEC-nIM-MS were in good agreement with nanoESI-nIM-MS values 24 . With therapeutic mAbs as model proteins, we compared CCS obtained with nanoESI-nIM-MS after manual desalting and SEC-nIM-MS analysis 28,33 . Different points should be considered when performing SEC-nIM-MS for CCS calculation.…”

“…While CIU appears as an elegant alternative to circumvent low IM resolution of linear TWIMS cells, lack of CIU automation in both buffer exchange and data acquisition has precluded its wide adoption. In order to fully automate CIU experiments from sample preparation to data interpretation, we have developed a fast-online SEC-CIU coupling using short SEC columns, where CVs are automatically raised along sample elution (Figure 4C) 33 . CIU fingerprints are generated with the CIUSuite 2 software to better visualize ATDs variations corresponding to conformational transitions (Figure 4D).…”

“…SEC afforded first a way to automate nMS experiments providing rapid assessment of proteins and complexes integrity of large numbers of samples, using small sample quantities (between 2 and 5 µg). The benefit of SEC-nMS has been broadly documented by our group 27,28,33 and others [29][30][31][32] for therapeutic proteins characterization, more particularly for mAb-based product analysis. In the present work, we aim at widening the scope of SEC-nMS use for different systems of interest we had in the lab.…”

Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins

“…8 However, CSF biomarker discovery is challenged by the low concentration of CSF proteins (as low as 0.15 mg/mL), 9 with CNS-specific proteins overshadowed by serum-derived proteins comprising ~40-80% (i.e., 0.06-0.12 mg/mL) of the CSF preprocessing methods 12 and can be used for removal of salts and detergents. [18][19][20][21][22] Notably, offline SEC can fractionate complex biological samples prior to RPLC-TDMS analysis, achieving a significant increase in protein/proteoform detection over RPLC-TDMS alone by not only reducing the sample complexity but also decreasing ion suppression of HMW proteins when present in the mass spectrometer with LMW proteins. 23,24 Online SEC-MS has been achieved, [25][26][27][28][29][30][31][32][33] but its widespread use, especially high resolution separations, has been precluded by a number of technical constraints.…”