title: An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells.
abstract: Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light-programmable, cell-permeable reporter deliverable simultaneously to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Emilie R
authors.initials: ER
authors.lastname: Mainz
authors.affiliation: Department of Chemistry, Division of Chemical Biology and Medicinal Chemistry and Department of Pharmacology, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Qunzhao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Division of Chemical Biology and Medicinal Chemistry and Department of Pharmacology, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Lawrence
authors.affiliation: Department of Chemistry and Pharmacology, University of North Carolina, Chapel Hill, NC, 27599, USA. nlallbri@unc.edu.|Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, Chapel Hill and Raleigh, NC, USA. nlallbri@unc.edu.
authors.forename: Nancy L
authors.identifier: 0000-0002-9242-768X
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D059010:Single-Cell Analysis
chemical_list: D051057:Proto-Oncogene Proteins c-akt
keywords: biosensors; chemical cytometry; kinase activity probes; photochemistry; single-cell analysis