Carter K. Asef, Jon Peraza, Casey E. Vantucci, Suraj Dhungana, and Mason Chilmonczyk
Andson Biotech, Atlanta, GA
Introduction
Native mass spectrometry (MS) is becoming increasingly valuable in modern biotherapeutics development. The study of proteins in their native state provides insights into higher-order structures, non-covalent interactions, and multimeric assemblies which are lost under denaturing conditions. Achieving high-quality nano-electrospray ionization (nanoESI) spectra for native MS remains challenging because buffers that preserve native conformations, such as Tris and PBS, are incompatible with MS. These buffers are difficult or time-consuming to remove, and insufficient cleanup leads to ion suppression, instrument contamination, and poor spectral quality. Consequently, gold-standard workflows rely on offline buffer exchange or size-exclusion chromatography (SEC) for cleanup, typically paired with passive nanoESI for sample introduction, significantly limiting overall throughput.
The DynaChip X1TM provides online buffer exchange and sample introduction in a single step to streamline native MS workflows. Coupling the DynaChipTM to an autosampler further increases throughput and reproducibility, and reduces the need for user intervention. Furthermore, programmable automated wash steps between runs minimize sample carryover. These capabilities are critical for high-volume applications such as formulation screening and cell-line development. Herein, we describe the coupling of the DynaChip X1TM to a Waters ACQUITY M-Class autosampler to enable automated, high-throughput native nanoESI workflows.
Experimental Methods
The DynaChip X1TM employs a microfabricated tangential flow mass exchanger for automated buffer exchange that can be used inline with nanoESI-MS. In this work, a DynaChip X1TM was placed inline between a Waters ACQUITY M-Class and a Waters Xevo G2-S QToF. The output line from the autosampler was connected directly to the injection port on the DynaChip X1TM using ZenFit connectors. Prior to running samples, the transit time from the autosampler to the end of the connecting line was characterized. This was accomplished by connecting the line directly to the MS and determining the time at which the injected sample was first detected. This characterization is necessary to establish appropriate timing parameters needed for autosampler integration with the DynaChip X1TM.
The DynaChipTM‘s method editor software allows users to define timed steps to control valve position and flow rates (Figure 1). Each step specifies a time point, a valve state (Load or Inject), and flow rates (for the syringe and the sample). The syringe flow rate represents the input flow rate, and the sample flow rate represents the output flow rate to the MS. During normal operation, these values are set equal; however, they are customizable to address special method needs. For this experiment, the method was configured so that the valve switched to Load at the appropriate time to capture the sample plug into the DynaChipTM sample loop as it arrived from the M-Class autosampler, then switched to Inject to deliver the sample to the MS at a flow rate of 0.5 µL/min. Between each sample injection, a wash step was programmed at an elevated flow rate of 1.5 µL/min to flush the system.
Figure 1: Image of the DynaChip X1TM Autosampler Method Editor. This screen allows users to create, save, and edit time-based methods that control valve position and flow rates. Steps can be added or removed as needed to accommodate different workflows. The user can define the number of samples to be processed, allowing the method to be repeated automatically across a full sample queue.
As a proof of concept, the platform was applied to the analysis of streptavidin (Thermo Fisher, Cat. #434301) and carbonic anhydrase II (Sigma Aldrich, Cat. #C7025). Ten injections of each protein at a concentration of 500 nM were performed, alternating between the two analytes, for a total of 20 injections. To minimize carryover, plugs of 30 mM n-octyl β-D-thioglucopyranoside (OTG; Anatrace, Cat. #O314), pH 7.5, were injected between each sample during the high flow rate wash step.
Results & Discussion
To evaluate the performance of the integrated autosampler-DynaChip X1TM workflow, 20 sequential injections were performed, alternating between streptavidin and carbonic anhydrase II, with wash buffer run between each sample. The results are summarized in Figure 2.
Figure 2: Integrated Autosampler and DynaChip X1TM Workflow for MS Analysis of Protein Standards. Extracted ion chromatograms (XIC) are shown for the 10 runs for both streptavidin (A) and carbonic anhydrase (B). The XICs for the first and last runs of the proteins (C) are shown next to the mass spectra that were averaged over the elution period (D). Minimal (<2%) to no carryover can be seen between the alternating protein runs.
Extracted ion chromatograms (XICs) for all 10 replicate injections of streptavidin (Figure 2A) and carbonic anhydrase II (Figure 2B) demonstrate relatively consistent elution profiles across the full run sequence. Peak shapes remained uniform from the first to the last injection for both analytes (Figure 2C), with no observable degradation in signal intensity or broadening over time. This consistency indicates that the automated injection and wash protocol maintained stable fluidic conditions throughout the experiment. The corresponding averaged mass spectra for the first and last runs of each protein (Figure 2D) show well-resolved charge state envelopes characteristic of native-like proteins, confirming that the inline buffer exchange provided by the DynaChip X1TM effectively removed non-volatile buffer components prior to nanoESI.
Critically, carryover between alternating protein injections was minimal, varying from no observed carryover to less than 2% carryover. This low level of cross-contamination demonstrates that the automated OTG wash step, performed at an elevated flow rate of 1.5 µL/min between each sample, was effective at clearing residual analyte from the system. Minimizing carryover is essential for applications where samples of varying identity or concentration are analyzed in sequence, such as formulation screening.
Each sample-to-sample cycle, including injection, elution, data acquisition, and the wash step, was completed in 20 minutes. While individual cycle times can vary depending on sample complexity and method parameters, this throughput represents a substantial improvement over traditional offline buffer exchange approaches, which often require manual desalting steps and passive nanoESI tip loading. The elimination of these manual steps not only increases throughput but also reduces operator-dependent variability, a key consideration for workflows requiring high reproducibility across large sample sets.
Taken together, these results demonstrate that the autosampler-DynaChip X1TM integration delivers reproducible, high-quality native mass spectra with minimal carryover and practical cycle times suitable for routine, high-throughput native protein analysis.
Conclusions
We have demonstrated successful integration of the DynaChip X1TM with a Waters ACQUITY M-Class autosampler, enabling reproducible sample processing with no significant carryover and consistent peak shape and intensity across replicates. Sample-to-sample analysis times of approximately 20 minutes, which includes the wash plug, represent a notable improvement in throughput compared to traditional offline buffer exchange approaches. The integration of the DynaChip X1™ with an autosampler provides a practical path toward scalable, automated native MS analysis for biotherapeutic development.
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LIT-AB-AS-26-001 _v1
