Ion Mobility-Mass Spectrometry Coupled to Droplet Microfluidics for Rapid Protein Structure Analysis and Drug Discovery

Abstract

Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by ∼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis.

Development of an Automated, High-Throughput Methodology for Native Mass Spectrometry and Collision-Induced Unfolding

“…We envision that nRapidFire-MS could become a transformative tool in the pharmaceutical sciences more generally as the methodology described herein can be used to collect high-throughput CIU data for a variety of therapeutic biomolecules such as mAbs and siRNA. RapidFire-CIU could also be leveraged in biopharma to conduct high-throughput drug-binding assays, wherein CIU could be deployed to quickly identify potential drug candidates and characterize them based on their mechanism of action. , Beyond biopharma, nRapidFire-MS could be of wide interest in structural biology research more generally by improving the throughput and efficiency of routine nMS analyses. nRapidFire-MS could even be deployed to facilitate the creation of a CIU database of the standard systems of interest.…”

“…RapidFire-CIU could also be leveraged in biopharma to conduct high-throughput drugbinding assays, wherein CIU could be deployed to quickly identify potential drug candidates and characterize them based on their mechanism of action. 17,32 Beyond biopharma, nRapidFire-MS could be of wide interest in structural biology research more generally by improving the throughput and efficiency of routine nMS analyses. nRapidFire-MS could even be deployed to facilitate the creation of a CIU database of the standard systems of interest.…”

“…28−31 In addition to nMS, recent efforts have been made to increase the throughput of CIU measurements, including droplet microfluidics for sample introduction and online size exclusion chromatography (SEC) that requires several minutes per sample. 29,30,32 In addition, recent advances in acoustic ejection-MS have allowed for MS experiments of significantly improved throughput targeting intact proteins; however, this technology has yet to be leveraged for nMS. 33 The RapidFire (RF) robotic system enables the automated, high-throughput analysis of samples from a well-plate format and performs online sample cleanup, with standard RapidFire-MS workflows utilizing solid phase extraction (SPE) with a reversed phase (RP) cartridge before eluting samples onto the MS instrument.…”

“…Multiple prior reports have described methods to improve the throughput of nMS. Examples include, the Advion Nanomate − and standard autosamplers coupled to fast desalting or separation technologies. − In addition to nMS, recent efforts have been made to increase the throughput of CIU measurements, including droplet microfluidics for sample introduction and online size exclusion chromatography (SEC) that requires several minutes per sample. ,, In addition, recent advances in acoustic ejection-MS have allowed for MS experiments of significantly improved throughput targeting intact proteins; however, this technology has yet to be leveraged for nMS …”

Collision Induced Unfolding Enables the Quantitation of Isomass Biotherapeutics in Complex Biological Matrices

“…Our data shows that the higher IM resolution offered by cIM can be instrumental in achieving stronger results with higher degrees of linearity. Prior reports have also demonstrated how nIM-MS can be adapted to enable high-throughput assays, with complete CIU fingerprints acquired in 30 s and with as little as a 40 nL consumed per sample, thus making it conceivable that quantitative CIU could be similarly automated and used in a high throughput fashion . Improving the throughput of quantitative CIU would be critically important for clinical assays in which quantitative CIU could be used to measure the concentrations of isomass proteins extracted from biological matrices.…”