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Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis

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Abstract

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6–464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times.

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References

  1. Brunelle JL, Green R. Chapter twelve — one-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol. 2014;541:151–9.

  2. Nowakowski AB, Wobig WJ, Petering DH. Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics. 2014;6(5):1068–78.

    Article  CAS  PubMed  Google Scholar 

  3. Gerver RE, Herr AE. Microfluidic western blotting of low-molecular-mass proteins. Anal Chem. 2014;86(21):10625–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bahga SS, Santiago JG. Coupling isotachophoresis and capillary electrophoresis: a review and comparison of methods. Analyst. 2013;138(3):735–54.

    Article  CAS  PubMed  Google Scholar 

  5. Wyckoff M, Rodbard D, Chrambach A. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: properties of the stack, valid Rf–measurement, and optimized procedure. Anal Biochem. 1977;78(2):459–82.

    Article  CAS  PubMed  Google Scholar 

  6. Schägger H. Tricine–SDS-PAGE. Nat Protoc. 2006;1(1):16–22.

    Article  PubMed  Google Scholar 

  7. Paratore F, ZeidmanKalman T, Rosenfeld T, Kaigala GV, Bercovici M. Isotachophoresis-based surface immunoassay. Anal Chem. 2017;89(14):7373–81.

    Article  CAS  PubMed  Google Scholar 

  8. Zhu Z, Lu JJ, Liu S. Protein separation by capillary gel electrophoresis: a review. Anal Chim Acta. 2012;709:21–31.

    Article  CAS  PubMed  Google Scholar 

  9. Bhilocha S, Amin R, Pandya M, Yuan H, Tank M, LoBello J, et al. Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan. Anal Biochem. 2011;417(1):41–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hermodson M. Current protocols in protein science, edited by J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield. New York: Wiley, 1995, 864 pages (core) + 576 pages (4 supplements)/year (updated looseleaf), print and CD-ROM. Proteins: Struct, Funct Bioinform. 1996;24(3):409-.

  11. Grabski A, Burgess R. Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips. Innovations. 2001;13:10–2.

    Google Scholar 

  12. Davis BJ. Disc electrophoresis. II. method and application to human serum proteins. Ann N Y Acad Sci. 1964;121:404–27.

    Article  CAS  PubMed  Google Scholar 

  13. Eubel H, Braun H-P, Millar A. Blue-native PAGE in plants: a tool in analysis of protein-protein interactions. Plant Methods. 2005;1(1):11.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Wittig I, Schägger H. Advantages and limitations of clear-native PAGE. Proteomics. 2005;5(17):4338–46.

    Article  CAS  PubMed  Google Scholar 

  15. Schägger H, Cramer WA, von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem. 1994;217(2):220–30.

    Article  PubMed  Google Scholar 

  16. PeliThanthri SH, Ward CL, Cornejo MA, Linz TH. Simultaneous preconcentration and separation of native protein variants using thermal gel electrophoresis. Anal Chem. 2020;92(9):6741–7.

    Article  CAS  Google Scholar 

  17. Crihfield CL, Holland LA. Protein sieving with capillary nanogel electrophoresis. Anal Chem. 2021;93(3):1537–43.

    Article  CAS  PubMed  Google Scholar 

  18. Mikšík I, Eckhardt A, Forgács E, Cserháti T, Deyl Z. The effect of sodium dodecyl sulfate and Pluronic F127 on the electrophoretic separation of protein and polypeptide test mixtures at acid pH. Electrophoresis. 2002;23(12):1882–6.

    Article  PubMed  Google Scholar 

  19. Wu D, Regnier FE. Native protein separations and enzyme microassays by capillary zone and gel electrophoresis. Anal Chem. 1993;65(15):2029–35.

    Article  CAS  PubMed  Google Scholar 

  20. Křížek T, Coufal P, Tesařová E, Sobotníková J, Bosáková Z. Pluronic F-127 as the buffer additive in capillary entangled polymer electrophoresis: some fundamental aspects. J Sep Sci. 2010;33(16):2458–64.

    Article  PubMed  Google Scholar 

  21. Rill RL, Al-Sayah MA. Peptide separations by slab gel electrophoresis in Pluronic F127 polymer liquid crystals. Electrophoresis. 2004;25(9):1249–54.

    Article  CAS  PubMed  Google Scholar 

  22. Wu X, Langan TJ, Durney BC, Holland LA. Thermally responsive phospholipid preparations for fluid steering and separation in microfluidics. Electrophoresis. 2012;33(17):2674–81.

    Article  CAS  PubMed  Google Scholar 

  23. Wu C, Liu T, Chu B. Viscosity-adjustable block copolymer for DNA separation by capillary electrophoresis. Electrophoresis. 1998;19(2):231–41.

    Article  CAS  PubMed  Google Scholar 

  24. Ward CL, Linz TH. Characterizing the impact of thermal gels on isotachophoresis in microfluidic devices. Electrophoresis. 2020;41(9):691–6.

    Article  CAS  PubMed  Google Scholar 

  25. Mikšík I, Sedláková P, Mikulíková K, Eckhardt A, Cserhati T, Horváth T. Matrices for capillary gel electrophoresis—a brief overview of uncommon gels. Biomed Chromatogr. 2006;20(6–7):458–65.

    Article  PubMed  Google Scholar 

  26. Zhang J, Gassmann M, He W, Wan F, Chu B. Reversible thermo-responsive sieving matrix for oligonucleotide separation. Lab Chip. 2006;6(4):526–33.

    Article  CAS  PubMed  Google Scholar 

  27. Jalaal M, Cottrell G, Balmforth N, Stoeber B. On the rheology of Pluronic F127 aqueous solutions. J Rheol. 2017;61(1):139–46.

    Article  CAS  Google Scholar 

  28. Svingen R, Åkerman B. Mechanism of electrophoretic migration of DNA in the cubic phase of Pluronic F127 and water. J Phys Chem B. 2004;108(8):2735–43.

    Article  CAS  Google Scholar 

  29. Ma J, Xia D. The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization. J Appl Crystallogr. 2008;41(Pt 6):1150–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cornejo MA, Linz TH. Harnessing Joule heating in microfluidic thermal gel electrophoresis to create reversible barriers for cell enrichment. Electrophoresis. 2021;42(11):1238–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cornejo MA, Linz TH. Multiplexed miRNA quantitation using injectionless microfluidic thermal gel electrophoresis. Anal Chem. 2022;94(14):5674–81.

    Article  CAS  PubMed  Google Scholar 

  32. Burton JB, Ward CL, Klemet DM, Linz TH. Incorporation of thermal gels for facile microfluidic transient isotachophoresis. Anal Methods. 2019;11(37):4733–40.

    Article  CAS  Google Scholar 

  33. Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N. Computer control of microscopes using µManager. Curr Protoc Mol Biol. 2010;92(1):14.20.1-14.20.17.

    Article  Google Scholar 

  34. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82.

    Article  CAS  PubMed  Google Scholar 

  35. Vaz FAS, Neves LNO, Marques R, Sato RT, Oliveira MAL. Chromophoreasy, an excel-based program for detection and integration of peaks from chromatographic and electromigration techniques. J Brazil Chem Soc. 2016;27:1899–911.

    CAS  Google Scholar 

  36. Haider SR, Sharp BL, Reid HJ. A comparison of tris-glycine and tris-tricine buffers for the electrophoretic separation of major serum proteins. J Sep Sci. 2011;34(18):2463–7.

    Article  CAS  PubMed  Google Scholar 

  37. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166(2):368–79.

    Article  PubMed  Google Scholar 

  38. Rath A, Cunningham F, Deber CM. Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts. Proc Natl Acad Sci USA. 2013;110(39):15668–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stellwagen NC. Apparent pore size of polyacrylamide gels: comparison of gels cast and run in tris-acetate-EDTA and tris-borate-EDTA buffers. Electrophoresis. 1998;19(10):1542–7.

    Article  CAS  PubMed  Google Scholar 

  40. Gallagher SR. One-dimensional electrophoresis using nondenaturing conditions. Curr Protoc Protein Sci. 2018;94(1): e73.

    Article  PubMed  Google Scholar 

  41. Durney BC, Lounsbury JA, Poe BL, Landers JP, Holland LA. A thermally responsive phospholipid pseudogel: tunable DNA sieving with capillary electrophoresis. Anal Chem. 2013;85(14):6617–25.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Financial support for this work was provided by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R21GM137278 and the National Science Foundation under Grant Number 2046487. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation. S.H.P.T. was supported by a Rumble fellowship from Wayne State University. The authors thank Prof. Mary Kay Pflum and Dr. Oscar McCrate for their assistance with protein labeling.

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  1. Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI, 48202, USA

    Shakila H. Peli Thanthri & Thomas H. Linz

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  1. Shakila H. Peli Thanthri
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Correspondence to Thomas H. Linz.

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry 2023 with guest editors Zhi-Yuan Gu, Beatriz Jurado-Sánchez, Thomas H. Linz, Leandro Wang Hantao, Nongnoot Wongkaew, and Peng Wu.

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Peli Thanthri, S.H., Linz, T.H. Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis. Anal Bioanal Chem 415, 4163–4172 (2023). https://doi.org/10.1007/s00216-022-04331-w

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  • DOI: https://doi.org/10.1007/s00216-022-04331-w

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