Abstract
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.
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References
Rodriguez-Merchan EC (2018) What’s new in gene therapy of hemophilia. Curr Gene Ther 18:107–114
Lykken EA, Shyng C, Edwards RJ, Rozenberg A, Gray SJ (2018) Recent progress and considerations for AAV gene therapies targeting the central nervous system. J Neurodev Disord 10:16
Bass-Stringer S, Bernardo BC, May CN, Thomas CJ, Weeks KL, McMullen JR (2018) Adeno-associated virus gene therapy: translational progress and future prospects in the treatment of heart failure. Heart Lung Circ 27(11):1285–1300
Allay JA, Sleep S, Long S, Tillman DM, Clark R, Carney G, Fagone P, McIntosh JH, Nienhuis AW, Davidoff AM, Nathwani AC, Gray JT (2011) Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther 22:595–604
Piras BA, Drury JE, Morton CL, Spence Y, Lockey TD, Nathwani AC, Davidoff AM, Meagher MM (2016) Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector. Mol Ther Methods Clin Dev 3:16015
Halder S, Van Vliet K, Smith JK, Duong TT, McKenna R, Wilson JM, Agbandje-McKenna M. (2015) Structure of neurotropic adeno-associated virus AAVrh.8. J Struct Biol 192:21–36
Eckard AD, Dupont DR, Young JK (2018) Development of two analytical methods based on reverse phase chromatographic and SDS-PAGE gel for assessment of deglycosylation yield in N-glycan mapping. Biomed Res Int 2018:3909674
Karageorgos I, Gallagher ES, Galvin C, Gallagher DT, Hudgens JW (2017) Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671. Biologicals 50:27–34
Voeten RLC, Ventouri IK, Haselberg R, Somsen GW (2018) Capillary electrophoresis: trends and recent advances. Anal Chem 90:1464–1481
Liu Y, Wang W, Jia M, Liu R, Liu Q, Xiao H, Li J, Xue Y, Wang Y, Yan C (2018) Recent advances in microscale separation. Electrophoresis 39:8–33
Breadmore MC, Wuethrich A, Li F, Phung SC, Kalsoom U, Cabot JM, Tehranirokh M, Shallan AI, Abdul Keyon AS, See HH, Dawod M, Quirino JP (2017) Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2014–2016). Electrophoresis 38:33–59
Chien RL, Burgi DS (1992) On-column sample concentration using field amplification in CZE. Anal Chem 64:489A–496A
Zhang CX, Thormann W (1996) Head-column field-amplified sample stacking in binary system capillary electrophoresis: a robust approach providing over 1000-fold sensitivity enhancement. Anal Chem 68:2523–2532
Zhang CX, Aebi Y, Thormann W (1996) Microassay of amiodarone and desethylamiodarone in serum by capillary electrophoresis with head-column field-amplified sample stacking. Clin Chem 42:1805–1811
Zhang CX, Thormann W (1998) Head-column field-amplified sample stacking in binary system capillary electrophoresis. 2. Optimization with a preinjection plug and application to micellar electrokinetic chromatography. Anal Chem 70:540–548
Song JZ, Chen J, Tian SJ, Sun ZPJ (1999) Assay for the determination of low dosage form of formoterol dry syrup by capillary electrophoresis with head-column field-amplified sample stacking. J Pharm Biomed Anal 21:569–576
Wey AB, Zhang CX, Thormann W (1999) Head-column field-amplified sample stacking in binary system capillary electrophoresis. Preparation of extracts for determination of opioids in microliter amounts of body fluids. J Chromatogr A 853:95–106
Chou YW, Huang WS, Chen CC, Lin SJ, Wu HL, Chen SH (2005) Trace analysis of zotepine and its active metabolite in plasma by capillary electrophoresis with solid phase extraction and head-column field-amplified sample stacking. J Chromatogr A 1087:189–196
Yang Y, Boysen RI, Hearn MT (2006) Optimization of field-amplified sample injection for analysis of peptides by capillary electrophoresis-mass spectrometry. Anal Chem 78:4752–4758
Ito E, Nakajima K, Waki H, Miseki K, Shimada T, Sato TA, Kakehi K, Suzuki M, Taniguchi N, Suzuki A (2013) Structural characterization of pyridylaminated oligosaccharides derived from neutral glycosphingolipids by high-sensitivity capillary electrophoresis-mass spectrometry. Anal Chem 85:7859–7865
Theurillat R, Sandbaumhüter FA, Bettschart-Wolfensberger R, Thormann W (2016) Microassay for ketamine and metabolites in plasma and serum based on enantioselective capillary electrophoresis with highly sulfated γ-cyclodextrin and electrokinetic analyte injection. Electrophoresis 37:1129–1138
Zhang CX, Meagher MM (2017) Sample stacking provides three orders of magnitude sensitivity enhancement in SDS capillary gel electrophoresis of adeno-associated virus capsid proteins. Anal Chem 89:3285–3292
Ciura K, Pawelec A, Buszewska-Forajta M, Markuszewski MJ, Nowakowska J, Prahl A, Wielgomas B, Dziomba S (2017) Evaluation of sample injection precision in respect to sensitivity in capillary electrophoresis using various injection modes. J Sep Sci 40:1167–1175
Wang Z, Guo H, Chen M, Zhang G, Chang R, Chen A (2018) Separation and determination of corynoxine and corynoxine B using chiral ionic liquid and hydroxypropyl-β-cyclodextrin as additives by field-amplified sample stacking in capillary electrophoresis. Electrophoresis 39(17):2195–2201. https://doi.org/10.1002/elps.201800129. Epub ahead of print
Šesták J, Thormann W (2017) Insights into head-column field-amplified sample stacking: Part I. Detailed study of electrokinetic injection of a weak base across a short water plug. J Chromatogr A 1502:51–61
Šesták J, Thormann W (2017) Insights into head-column field-amplified sample stacking: Part II. Study of the behavior of the electrophoretic system after electrokinetic injection of cationic compounds across a short water plug. J Chromatogr A 1512:124–132
Šesták J, Theurillat R, Sandbaumhüter FA, Thormann W (2018) Fundamental aspects of field-amplified electrokinetic injection of cations for enantioselective capillary electrophoresis with sulfated cyclodextrins as selectors. J Chromatogr A 1558:85–95
Acknowledgements
Dr. Timothy Lockey, Dr. Bryan Piras, Dr. Jason Drury, Dr. Susan Sleep, and Dr. Clifford Froelich are greatly acknowledged for providing AAV samples and analytical support. This work was supported by American Lebanese Syrian Associated Charities (ALSAC).
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Zhang, CX., Meagher, M.M. (2019). Highly Sensitive SDS Capillary Gel Electrophoresis with Sample Stacking Requiring Only Nanograms of Adeno-Associated Virus Capsid Proteins. In: Phillips, T.M. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology, vol 1972. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9213-3_19
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DOI: https://doi.org/10.1007/978-1-4939-9213-3_19
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