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Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions

  • Claire M. Ouimet
  • Cara I. D’Amico
  • Robert T. KennedyEmail author
Research Paper
  • 29 Downloads

Abstract

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device.

Graphical abstract

Keywords

Microfluidics/microfabrication Capillary electrophoresis/electrophoresis Proteins Droplets 

Notes

Acknowledgments

The authors gratefully acknowledge Jason Gestwicki, Hao Shao, and Jennifer Rauch of the University of California-San Francisco for providing inhibitors and proteins used in this work.

Funding information

This work was financially supported by NIH R01GM102236 (RTK), NIH T32-GM007767 (CID), and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship sponsored by Eli Lilly and Company (CMO).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2006_MOESM1_ESM.pdf (338 kb)
ESM 1 (PDF 337 kb)

References

  1. 1.
    Ouimet CM, D’Amico CI, Kennedy RT. Advances in capillary electrophoresis and the implications for drug discovery. Expert Opin Drug Discov [Internet]. Taylor & Francis. 2017;12(2):213–24.CrossRefGoogle Scholar
  2. 2.
    Perrin D, Frémaux C, Shutes A. Capillary microfluidic electrophoretic mobility shift assays: application to enzymatic assays in drug discovery. Expert Opin Drug Discov [Internet]. 2009;15:51–63.Google Scholar
  3. 3.
    Wells JA, McClendon CL. Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature [Internet]. 2007;450(7172):1001–9.CrossRefGoogle Scholar
  4. 4.
    Rauch JN, Nie J, Buchholz TJ, Gestwicki JE, Kennedy RT. Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions. Anal Chem. 2013;85(20):9824–31.CrossRefGoogle Scholar
  5. 5.
    Perrin D, Frémaux C, Scheer A. Assay development and screening of a serine/threonine kinase in an on-chip mode using caliper nanofluidics technology. J Biomol Screen [Internet]. 2006;11(4):359–68.CrossRefGoogle Scholar
  6. 6.
    Jacobson SC, Hergenroder R, Koutny LB, Ramsey JM. High-speed separations. Anal Chem. 1994;66(7):1114–8.CrossRefGoogle Scholar
  7. 7.
    Lo CT, Throckmorton DJ, Singh AK, Herr AE. Photopolymerized diffusion-defined polyacrylamide gradient gels for on-chip protein sizing. Lab Chip. 2008;8(8):1273–9.CrossRefGoogle Scholar
  8. 8.
    Roman GT, Wang M, Shultz KN, Jennings C, Kennedy RT. Sampling and electrophoretic analysis of segmented flow streams using virtual walls in a microfluidic device. Anal Chem. 2008;80(21):8231–8.CrossRefGoogle Scholar
  9. 9.
    Wang M, Roman GT, Perry ML, Kennedy RT. Microfluidic chip for high efficiency electrophoretic analysis of segmented flow from a microdialysis probe and in vivo chemical monitoring. Anal Chem. 2009;81(21):9072–8.CrossRefGoogle Scholar
  10. 10.
    Niu XZ, Zhang B, Marszalek RT, Ces O, Edel JB, Klug DR, et al. Droplet-based compartmentalization of chemically separated components in two-dimensional separations. Chem Commun (Camb) [Internet]. 2009;(41):6159–61.Google Scholar
  11. 11.
    Pei J, Nie J, Kennedy RT. Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities. Anal Chem. 2010;82(22):9261–7.CrossRefGoogle Scholar
  12. 12.
    Guetschow ED, Steyer DJ, Kennedy RT. Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators. Anal Chem. 2014;86:10373–9.CrossRefGoogle Scholar
  13. 13.
    Delamarre MF, Shippy SA. Development of a simplified microfluidic injector for analysis of droplet content via capillary electrophoresis. Anal Chem. 2014;86:10193–200.CrossRefGoogle Scholar
  14. 14.
    Edgar JS, Pabbati CP, Lorenz RM, He M, Fiorini GS, Chiu DT. Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis. Anal Chem. 2006;78(19):6948–54.CrossRefGoogle Scholar
  15. 15.
    Austin C, Pettit SN, Magnolo SK, Sanvoisin J, Chen W, Wood SP, et al. Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors. J Biomol Screen. 2012;17(7):868–76.CrossRefGoogle Scholar
  16. 16.
    Niu X, Pereira F, Edel JB, Demello AJ. Droplet-interfaced microchip and capillary electrophoretic separations. Anal Chem. 2013;85(18):8654–60.CrossRefGoogle Scholar
  17. 17.
    Li Q, Zhu Y, Zhang N-Q, Fang Q. Automatic combination of microfluidic nanoliter-scale droplet array with high-speed capillary electrophoresis. 2016;6:26654.Google Scholar
  18. 18.
    Guetschow ED, Kumar S, Lombard DB, Kennedy RT. Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples. Anal Bioanal Chem [Internet]. 2016:1–11.Google Scholar
  19. 19.
    Ouimet CM, Dawod M, Grinias J, Assimon VA, Lodge J, Mapp AK, et al. Protein cross-linking capillary electrophoresis at increased throughput for a range of protein – protein interactions. Analyst Royal Society of Chemistry. 2018;143:1805–12.Google Scholar
  20. 20.
    Ouimet CM, Shao H, Rauch JN, Dawod M, Nordhues BA, Dickey CA, et al. Protein cross-linking capillary electrophoresis for protein-protein interaction analysis. Anal Chem. 2016;88(16):8272–8.CrossRefGoogle Scholar
  21. 21.
    Nagata H, Tabuchi M, Hirano K, Baba Y. High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution. Electrophoresis. 2005;26:2687–91.CrossRefGoogle Scholar
  22. 22.
    Harrison DJ, Fluri K, Seiler K, Fan Z, Effenhauser CS, Manz A. Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip. Science (80- ). 1993;261(5123):895–7.CrossRefGoogle Scholar
  23. 23.
    Roper MG, Shackman JG, Dahlgren GM, Kennedy RT. Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay. Anal Chem. 2003;75(18):4711–7.CrossRefGoogle Scholar
  24. 24.
    Simpson PC, Woolley AT, R a M. Microfabrication technology for the production of capillary array electrophoresis chips. Biomed Microdevices. 1998;1(12):7–26.CrossRefGoogle Scholar
  25. 25.
    Shackman JG, Watson CJ, Kennedy RT. High-throughput automated post-processing of separation data. J Chromatogr A. 2004;1040(2):273–82.CrossRefGoogle Scholar
  26. 26.
    Chabert M, Dorfman KD, De Cremoux P, Roeraade J, Viovy JL. Automated microdroplet platform for sample manipulation and polymerase chain reaction. Anal Chem. 2006;78(22):7722–8.CrossRefGoogle Scholar
  27. 27.
    Li X, Srinivasan SR, Connarn J, Ahmad A, Young ZT, Kabza AM, et al. Analogues of the allosteric heat shock protein 70 (Hsp70) inhibitor, MKT-077, as anti-cancer agents. ACS Med Chem Lett. 2013;4(11):1042–7.CrossRefGoogle Scholar
  28. 28.
    Shao H, Li X, Moses MA, Gilbert LA, Kalyanaraman C, Young ZT, et al. Exploration of benzothiazole rhodacyanines as allosteric inhibitors of protein−protein interactions with heat shock protein 70 (Hsp70). 2018;70.Google Scholar
  29. 29.
    Koren J, Miyata Y, Kiray J, O’Leary JC, Nguyen L, Guo J, et al. Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance. PLoS One. 2012;7(4).Google Scholar
  30. 30.
    Bousse L, Mouradian S, Minalla a, Yee H, Williams K, Dubrow R. Protein sizing on a microchip. Anal Chem. 2001;73(6):1207–12.CrossRefGoogle Scholar
  31. 31.
    Du J, Zhou Y, Su X, Yu JJ, Khan S, Jiang H, et al. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science (80). 2011;334(6057):806–9.CrossRefGoogle Scholar
  32. 32.
    Park J, Chen Y, Tishkoff DX, Peng C, Tan M, Dai L, et al. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. Mol Cell Elsevier Inc. 2013;50(6):919–30.CrossRefGoogle Scholar
  33. 33.
    Lu W, Zuo Y, Feng Y, Zhang M. SIRT5 facilitates cancer cell growth and drug resistance in non-small cell lung cancer. Tumor Biol. 2014:10699–705.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Claire M. Ouimet
    • 1
  • Cara I. D’Amico
    • 2
  • Robert T. Kennedy
    • 1
    • 2
    Email author
  1. 1.Department of ChemistryUniversity of MichiganAnn ArborUSA
  2. 2.Department of PharmacologyUniversity of MichiganAnn ArborUSA

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