DIGITAL MICROFLUIDIC CHIPS FOR AUTOMATED HYDROGEN DEUTERIUM EXCHANGE (HDX) MS ANALYSIS

L. Zhao1,2,3, C. M. Ryan4,5, K. Liu1,2,3,6, K. F. Faull4,5, J. Whitelegge4,5, C. K.-F. Shen1,2,3 1Crump Institute for Molecular Imaging, David Geffen School of Medicine at University of California Los Angeles, USA, 2California Nanosystems Institute at University of California Los Angeles, USA, 3Department of Molecular and Medical

Pharmacology, David Geffen School of Medicine at University of California Los Angeles, USA, 4The Pasarow Mass Spectrometry Laboratory, David Geffen School of Medicine at University of California Los Angeles, USA, 5Semel Institute for Neuroscience, David Geffen School of Medicine at University of California Los Angeles, USA, 6College of Electronics

and Information Engineering, Wuhan Textile University, Wuhan, People’s Republic of China ABSTRACT

We have developed a digital microfluidic droplet generator (DMDG) chip that provides an automated platform for generating nanoliter droplets with precisely defined compositions pre-programmed by the user. Coupling with an aqueous-organic micro-size-exclusion chromatography (µ-SEC) system and an HPLC injector, chip-based hydrogen/deuterium exchange (HDX) experiments with model proteins, myoglobin (Mb) and bacteriorhodopsin (bR), were performed and yielded the corresponding protein mass spectra within 40 seconds after fast SEC chromatography. This microfluidic platform could potentially overcome the hurdles of current HDX operations, and provide a practical solution for those who want to utilize HDX-MS for routine protein characterizations. KEYWORDS: Digital microfluidics, hydrogen/deuterium exchange (HDX), protein structure, mass spectrometry INTRODUCTION

Mass-based hydrogen/deuterium exchange (HDX-MS) experiments probe protein structures by monitoring the rate and extent of deuterium exchange with backbone amide protons. This approach has proven to be a useful method for documenting protein dynamics and changes to protein conformation in solution [1,2]. There are many important applications of HDX-MS, including characterization of proteins that are used as therapeutic/pharmacological agents (QA/QC of protein therapeutics), real-time visualization of protein-protein/protein-ligand interactions (for studying signaling pathways before and after drug perturbation), structure determination of proteins in solution (other than in solid-state crystal), screening ligands that can interact or stabilize certain proteins (for drug discovery and selecting ligands that can facilitate protein crystallization for X-ray crystallographic characterization), to name a few. However, the HDX process is sensitive to environmental factors (temperature, pH, salt concentration, detergents, etc.) as well as other experimental and temporal logistics. In addition, the biggest problem of HDX experiments is the fast D/H back-exchange (from D back to H after the initial HDX has been quenched), especially during the final chromatographic separation with non-deuterated solvents. As a consequence, different degrees of back-exchange result in loss of pertinent data, low reproducibility and also confound data analysis. An automated platform, which can reliably manipulate small amounts of sample to rapidly perform HDX and be interfaced with current HPLC-ESI-MS systems, will be highly desirable. Integral membrane proteins present one third of the cellular proteome and include many potential therapeutic targets. However, analyzing these proteins using HDX-MS is challenging due to the hydrophobic transmembrane domains that limit their solubility in aqueous solvents. In addition, the HDX process is sensitive to environmental factors as well as operation duration and sequences. Therefore low reproducibility of such experiments is often observed. Technical improvements that improve the handling of integral membrane proteins and help the analysis of transmembrane domains are therefore of importance. Our goal is to develop a reliable microfluidic-based platform capable of performing automated HDX operation on-chip and interfacing with ESI-MS for immediate sample analysis. THEORY

A typical HDX experiment begins by dilution of a protein solution at least eight- to ten-fold with D2O buffer. After each of a series of H/D exchange periods, the reaction is quenched by lowering the pH to about 2.3–2.5 (usually by addition of formic acid) and temperature to about 0 °C (to prevent back-exchange). For top-down analysis, the sample is directly injected into HPLC for separation and then into ESI-MS for detection. A very rapid on-line high performance liquid chromatography (HPLC) step prepares the sample for ESI-MS analysis. With increasing H/D exchange period, the mass of each exposed segment of the protein increases, and can be monitored by MS. Since protein function is dictated by protein conformation, the relationship between isotopic exchange rates of main chain amide hydrogens in proteins and their secondary and tertiary structures can be used to distinguish their structures in solution. Therefore, HDX-MS has proven to be an extremely useful analytical method for the study of protein dynamics and changes to protein conformation.

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 1287 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

EXPERIMENTAL Protein solution, detergent, H2O and D2O were first loaded into small glass vials and transferred under positive N2

pressure via PTFE microbore lines directly into the corresponding inlets on our DMDG chip (see Figure 1 & [3] for detail). After priming all the lines, the DMDG chip was washed with H2O (3-5 µL) and D2O (1-3 µL) and then dried with a stream of N2 before each experiment. Microvalves were used to isolate each reagent/sample inlet so that the incoming reagents are not in contact with sample until the moment of droplet formation. They will be rapidly mixed and reacted thereafter while moving along the microfluidic channel. As a proof-of-concept prototype, we applied a 1-mL disposable pipette tip, which served as a reservoir for product collection. As droplets coming out of the microbore tube, they were sprayed out and stuck on the tip due to surface tension. At the end of experiment, a fixed volume of formic acid (about 24 µL) was delivered to the same tip. Finally the entire solution was mixed thoroughly by bubbling with N2 and loaded into the sample loop ready for HPLC injection. The rapid HPLC separation was carried out at 50 µL/min with a custom-made, miniaturized size exclusion column (1mm x 5cm, filled with Tosoh TSK-gel SW2000) using elution buffer containing chloroform, methanol, 1% formic acid in H2O (4:4:1, v/v/v). The eluent was directed to the electrospray ionization source of the ESI-MS. The mass spectra were record on either a 7T Thermo Scientific LTQ-FT Ultra hybrid linear ion trap/FTICR mass spectrometer or a Perkin Elmer Sciex API III triple quadrupole mass spectrometer fitted with ion spray source. The mass spectrometer were tuned and calibrated before HDX experiment by flow injection of a mixture of polyproylene glycol (PPG). After injection, the first peak containing a fraction of deuterated protein generally showed up around 30-40 seconds.

Figure 1: Schematic illustration of DMDG chip & micro-size-exclusion chromatography system (µ-SEC) for HDX-MS. RESULTS & DISCUSSION

The DMDG Chip [3-5] is an automated microfluidic platform that generates nanoliter droplets with well-defined compositions preprogrammed by the user (Figure 1). Since only small amounts of sample is required, it has been used to scout for optimal radiolabeling conditions of proteins and peptides, and to screen for the optimal combination of individual building blocks for assembling supramolecular nanoparticles (SNPs) with tailored biological properties. Herein, we extend the application of the DMDG device and report successful multi-step HDX experiments. The biggest problem using HDX-MS to probe protein structures is the back-exchange. Different degrees of back-exchange result in loss of pertinent data and also confound data analysis. Therefore our first experiment was designed to see the extent of back-exchange using a DMDG chip. Myoglobin (Mb), an iron- and oxygen-binding protein found in the muscle tissue in almost all mammals, was chosen as a model system. Mb (3 mg/mL) and deuterated formic acid (D-FA) was mixed in the chip with a volumetric ratio of 1:10. A 24 Da increase in mass was observed which represents the extent of back-exchange during the quenching step. Next, we carried out HDX experiments by incubating Mb with (i) D2O then quenched with FA, representing the full forward exchange, and (ii) D2O then quenched with D-FA, representing the full forward exchange without back-exchange during quenching. The observed mass shifts compared to pristine Mb were recorded as +46.3 Da for (i) and +84.4 Da for (ii), respectively (Figure 2). A number of different configurations were investigated using myoglobin as well as an integral membrane protein, bacteriorhodopsin (bR), as standards (data not shown). Optimization of loop volume, column geometry and flow rate has improved the separation efficiency that could yield protein mass spectra within 40 seconds with speedy column regeneration (< 5 minutes), for protein loads in the sub-microgram range for both standards using the 1 mm column. One immediate advantage afforded by the rapidity of the separation relates to preservation of labile post-translational modifications. The model integral membrane protein bR has a retinal chromophore that becomes susceptible to hydrolysis under the semi-denaturing conditions used such that around 50% is lost after the 8-10 minutes it takes to elute the protein

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from the classic 4.6 x 300 mm column. With the 40-second µ-SEC method we observed practically no hydrolysis of the chromophore. Obviously there are tremendous throughput benefits as well moving from 2 samples/hour previously to 12 or more per hour using DMDG chip and µ-SEC system. Currently we are testing how detergent can affect the structure of bR in solution using HDX-MS.

Figure 2: On-chip HDX analyses of myoglobin (Mb) using µ-SEC system and ESI-MS.

CONCLUSION

In summary, we have successfully demonstrated chip-based HDX experiments using on-line ESI-MS. This microfluidic-assisted HDX system provides several important advantages over other technologies or platforms, such as (1) sample economy; (2) rapid mixing and processing; (3) constant and controllable environment; (4) automated and programmable operations; (5) scalable for high-throughput; (6) modular and flexible design. We have performed highly reproducible HDX operations with a common soluble protein, myoglobin, and a model integral membrane protein, bacteriorhodopsin (bR)[6] (data not shown), using DMDG chips and generated high-resolution mass spectrometric data using an on-line FT-ICR ESI-MS. A versatile design allows the DMDG chip to be easily interfaced with any type of mass spectrometer, and presents an ideal technological platform that adopts all the requirements to make HDX MS-based biological assay a routine process. ACKNOWLEDGEMENTS

This study was supported by the US Department of Energy (DE-FG02-09ER09-08 and DE-PS02-09ER09-18), and the Industry–University Cooperative Research Program (UC Discovery Grant, bio07-10665). We thank Dr. James Bowie and Dr. Cao Zheng in Department of Chemistry and Biochemistry at UCLA to provide bacteriorhodopsin sample. We thank Dr. Melissa Sondej and Dr. Rudy Alvarado in UCLA Molecular Instrumentation Center (MIC) to provide assistance for FT-ICR MS. REFERENCES [1] K. F. Faull, J. Higginson, A. J. Waring, T. To, J. P. Whitelegge, R. L. Stevens, C. B. Fluharty, A. L. Fluharty, The

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[6] S. Faham, G. L. Boulting, E. A. Massey, S. Yohannan, D. Yang, J. U. Bowie, Crystallization of bacteriorhodopsin from bicelle formulations at room temperature, Protein Sci, 14(3), 836-840 (2005).

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