Abstract
Dysregulation of cytokine signaling pathways is associated with benign and malignant hematologic disorders. Improvements in therapy rely on understanding the biology of the pathways and the proteins involved. Studying these pathways in patient samples is challenging as samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. To address some of these difficulties, we have utilized the technique of microcapillary electrophoresis. Using the NanoPro 1000 system (ProteinSimple) which is built on an automated, capillary-based immunoassay platform, we have developed rapid and quantitative assays for specific proteins from relatively small sample sizes. The NanoPro provides precise and quantitative data of the phosphorylation states of a specific protein of interest. We describe our experience with NanoPro assay development and optimization with specific application toward understanding aberrant cytokine signaling in human leukemia cells.
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References
Kalaitzidis D, Neel BG (2008) Flow-cytometric phosphoprotein analysis reveals agonist and temporal differences in responses of murine hematopoietic stem/progenitor cells. PLoS One 3:e3776
Han L, Wierenga AT, Rozenveld-Geugien M, van de LK, Vellenga E, Schuringa JJ (2009) Single-cell STAT5 signal transduction profiling in normal and leukemic stem and progenitor cell populations reveals highly distinct cytokine responses. PLoS One 4:e7989
Bendall SC, Simonds EF, Qiu P, Amir E, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe’er D, Tanner SD, Nolan GP (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332:687–696
Wong SC, Chan CM, Ma BB, Lam MY, Choi GC, Au TC, Chan AS, Chan AT (2009) Advanced proteomic technologies for cancer biomarker discovery. Expert Rev Proteomics 6:123–134
Knittle JE, Roach D, Horn PB, Voss KO (2007) Laser-induced fluorescence detector for capillary-based isoelectric immunoblot assay. Anal Chem 79:9478–9483
O’Neill RA, Bhamidipati A, Bi X, Deb-Basu D, Cahill L, Ferrante J, Gentalen E, Glazer M, Gossett J, Hacker K, Kirby C, Knittle J, Loder R, Mastroieni C, Maclaren M, Mills T, Nguyen U, Parker N, Rice A, Roach D, Suich D, Voehringer D, Voss K, Yang J, Yang T, Vander Horn PB (2006) Isoelectric focusing technology quantifies protein signaling in 25 cells. Proc Natl Acad Sci U S A 103:16153–16158
Fan AC, Deb-Basu D, Orban MW, Gotlib JR, Natkunam Y, O’Neill R, Padua RA, Xu L, Taketa D, Shirer AE, Beer S, Yee AX, Voehringer DW, Felsher DW (2009) Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat Med 15:566–571
Chen JQ, Lee JH, Herrmann MA, Park KS, Heldman MR, Goldsmith PK, Wang Y, Giaccone G (2013) Capillary isoelectric-focusing immunoassays to study dynamic oncoprotein phosphorylation and drug response to targeted therapies in non-small cell lung cancer. Mol Cancer Ther 12:2601–2613
Iacovides DC, Johnson AB, Wang N, Boddapati S, Korkola J, Gray JW (2013) Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay. Mol Cell Proteomics 12:3210–3220
Kentsis A, Reed C, Rice KL, Sanda T, Rodig SJ, Tholouli E, Christie A, Valk PJ, Delwel R, Ngo V, Kutok JL, Dahlberg SE, Moreau LA, Byers RJ, Christensen JG, Vande WG, Licht JD, Kung AL, Staudt LM, Look AT (2012) Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia. Nat Med 18:1118–1122
Espina V, Edmiston KH, Heiby M, Pierobon M, Sciro M, Merritt B, Banks S, Deng J, VanMeter AJ, Geho DH, Pastore L, Sennesh J, Petricoin EF III, Liotta LA (2008) A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Mol Cell Proteomics 7:1998–2018
Acknowledgements
The authors thank the Aflac Cancer and Blood Disorders Center Leukemia/Lymphoma Research Program, Children’s Healthcare of Atlanta, the Emory + Children’s Pediatric Flow Cytometry Core Facility, and the Cure Childhood Cancer Foundation for supporting the NanoPro assay development.
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Bradley, H.L., Sabnis, H., Pritchett, D., Bunting, K.D. (2014). Nanoproteomic Assays on Hematopoietic Stem Cells. In: Bunting, K., Qu, CK. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Biology, vol 1185. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1133-2_11
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DOI: https://doi.org/10.1007/978-1-4939-1133-2_11
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-1133-2
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